# Patent

## Title
Genetically modified plants with resistance to Xanthomonas and other bacterial plant pathogens

## Abstract

Applicants have successfully generated heritable phenotypes in plants making them resistant to bacterial blight. TAL effector binding elements, (EBEs) of bacterial pathogen disease susceptibility genes are modified to prevent induction of expression associated with disease states caused by the bacterial pathogens. Surprisingly, Applicants have found that modifications may be made in the EBEs of these genes which prevent bacterial pathogen induction, but still allow for normal plant development and seed production. Nucleic acid sequences for generating such plants, amino acid sequences, cells, vectors and expression constructs are included as well as resistant plants, seeds and lines.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a National Phase application claiming priority to PCT/US2012/71722 filed Dec. 27, 2012 which claims priority under 35 U.S.C. §119 to provisional application U.S. Ser. No. 61/581,356 filed Dec. 28, 2011, all of which are herein incorporated by reference in their entireties.


GRANT REFERENCE
This invention was made with government support under Grant No. DBI-0820831 awarded by US National Science Foundation. The government has certain rights in the invention.


TECHNICAL FIELD
The invention relates to the control of pathogens. Disclosed herein are methods of producing transgenic plants with increased pathogen resistance, mutated pathogen target sequences, that prevent pathogen activation but retain activity for normal plant development, polynucleotides for engineering the same, and transgenic plants and seeds generated therefrom.
BACKGROUND OF THE INVENTION
Bacterial blight of rice, caused by the pathogenic bacterium Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating rice diseases, often resulting in yield losses of up to 50% and sometimes destroying an entire crop. The outcome of this disease is largely dictated by a few transcription activator-like (TAL) effectors that are secreted from bacteria into host rice cells. These TAL effectors bind to endogenous promoters and activate expression of corresponding disease susceptibility (S) genes. Such TAL effectors are essentially virulence factors of the pathogenic strains, and the host S gene induction is required for the plant to become susceptible to the disease. Thus the components of this disease complex represent a target for intervention. Complicating intervention strategies, the S genes are critical to plant survival and play an important role in growth and development in rice, making typical knockout strategies inappropriate.
The known TAL effectors of essential virulence include PthXo1, AvrXa7, PthXo3, and PthXo2 in a variety of Xoo strains, while the corresponding S genes are s11N3, Os8N3, and Os12N3 of the N3 family of rice. Naturally occurring genetic variations within the promoter regions of Os8N3 and Os12N3 prevent their inducibility and have been found to confer resistance to the strains that depend on the respective PthXo1 and PthXo2 for virulence in the otherwise susceptible rice cultivars. There is no known genetic variation in Os11N3 that confers disease resistance to AvrXa7 and PthXo3-dependant Xoo strains.
TAL effector nucleases (TALENs), fusion proteins of the DNA cleavage domain of endonuclease FokI and the various forms of TAL effectors (native or custom-made, truncated or complete), have quickly emerged as efficient endonucleases to direct double-strand DNA breaks and induce genetic alterations at pre-selected loci in presumably any eukaryotic organism.
A need exists in the art for plants that are resistant to Xanthomonas oryzae pv. oryzae (Xoo).
It is an object of the present invention to provide mutations in the Os11N3 promoter region that cause resistance to Xoo while still retaining plant growth and plant reproduction.
It is further object of the present invention to provide mutations in the promoter regions of other S genes, either individually or tandemly, that cause resistance to Xoo while still retaining plant growth and plant reproduction.
SUMMARY OF THE INVENTION
According to the invention, Applicants have successfully generated heritable phenotypes in plants making them resistant to bacterial blight. TAL effector binding elements (EBEs) of bacterial pathogen disease susceptibility genes which are targeted by TAL effectors of essential virulence are modified to prevent induction of expression associated with disease states caused by the bacterial pathogens. Surprisingly, Applicants have found that modifications may be made in the EBEs of these genes which prevent bacterial pathogen induction, but still allow for normal plant development and seed production.
In one embodiment, EBE regions of the N3 family of genes in rice are modified with deletions or insertions while the TATAAA box region remains intact. This includes but is not limited to Os11N3 (EBE SEQ ID NO:16 (−258 to −231 from the Os11N3 ATG start site) and SEQ ID NO:17 (−260 to −232 from the Os11N3 ATG start site), Os8N3 (EBE SEQ ID NO:19), and Os12N3 (EBE SEQ ID NO:18) of the N3 family of rice. According to the invention the EBEs are modified so that TAL effectors of essential virulence PthXo1, AvrXa7, PthXo3, and PthXo2 are prevented from induction of expression, while plant growth and seed development are retained.
In one embodiment, Applicants have identified and created modifications in the overlapping PthXo3 and AvrXa7 EBEs of Os11N3 that inhibit the induction by AvrXa7 and PthXo3, and yet surprisingly plants with these modifications still exhibit normal plant growth and seed production. The overlapping portions (SEQ ID NO:20) of the two EBEs which do not include the TATAAA box are thus targeted and subjected to modification according to the invention.
In one embodiment, the invention includes a modified Os11N3 EBE nucleic acid sequence of SEQ ID NOS: 5, 6, 7, or 9 which provides resistance to Xoo-mediated induction and bacterial blight, but which also maintains normal plant growth and development. SEQ ID NO:5 is a deletion of 9 nucleotides from wild type SEQ ID NO:1, bases 27-35, SEQ ID NO: 6 is a deletion of 5 nucleotides or bases 27-31 from wild type SEQ ID NO:1, SEQ ID NO: 7 is a deletion of 4 nucleotides or bases 34-38 from wild type SEQ ID NO:1, and SEQ ID NO: 9 is an insertion of 9 bases (gtttatata) between bases 37 and 38 from wild type SEQ ID NO:1.
In another embodiment, a nucleotide sequence that is at least 90 or greater, 95% or greater percent similar to SEQ ID NO: 5, 6, 7, or 9 which still inhibits AvrXa7 or PthXo3 induction yet retains normal plant development. In yet another embodiment, a nucleotide sequence that hybridizes to SEQ ID NO 5, 6, 7, or 9 under stringent hybridization conditions involving, for example, hybridization in a hybridization buffer containing, for example, 20 percent formamide in 0.9M saline/0.09M SSC buffer, at a temperature of about 42° C. which still inhibits AvrXa7 or PthXo3 induction yet retains normal plant development are also within the scope of the invention. The invention also includes, plants, cells, seeds, plant tissues and the like which include this modification.
The present invention also relates to a method of imparting bacterial blight resistance to plants. This method involves providing a plant or plant seed comprising a modified EBE such as SEQ ID NO:5, 6, 7, or 9 and growing the modified plant or a plant produced from the modified plant seed under conditions effective to impart resistance to the plant. The present invention further relates to plants produced by this method, as well as component parts, seeds, and fruits of the plant.
In one embodiment, the invention includes a modified Os08N3 EBE nucleic acid sequence of SEQ ID NOS: 44, 45, 46, or 47 which provides resistance to Xoo-mediated induction and bacterial blight, but which also maintains normal plant growth and development. SEQ ID NO:44 is a deletion of 6 nucleotides from wild type SEQ ID NO:44, 65 to 60 bases upstream of the transcription initiation site (bases 22 through 27 of SEQ ID NO:43); SEQ ID NO: 45 is a deletion of 8 nucleotides or bases 22-29 from wild type SEQ ID NO:43; SEQ ID NO: 46 is a deletion of 21 nucleotides or bases 16-36 from wild type SEQ ID NO:43 with the base at position 15 changed to a thymine from a cytosine in wild type SEQ ID NO:43; and SEQ ID NO: 47 is a deletion of 14 nucleotides or bases 16-30 from wild type SEQ ID NO:43.
In another embodiment, a nucleotide sequence that is at least 90 or greater, 95% or greater percent similar to SEQ ID NO: 44, 45, 46, or 47 which still inhibits AvrXa7 or PthXo3 induction yet retains normal plant development. In yet another embodiment, a nucleotide sequence that hybridizes to SEQ ID NO: 44, 45, 46, or 47 under stringent hybridization conditions involving, for example, hybridization in a hybridization buffer containing, for example, 20 percent formamide in 0.9M saline/0.09M SSC buffer, at a temperature of about 42° C. which still inhibits AvrXa7 or PthXo3 induction yet retains normal plant development are also within the scope of the invention. The invention also includes, plants, cells, seeds, plant tissues and the like which include this modification.
The present invention also relates to a method of imparting bacterial blight resistance to plants. This method involves providing a plant or plant seed comprising a modified EBE such as SEQ ID NO: 44, 45, 46, or 47 and growing the modified plant or a plant produced from the modified plant seed under conditions effective to impart resistance to the plant. The present invention further relates to plants produced by this method, as well as component parts, seeds, and fruits of the plant.
In addition the present invention is directed to transferring the modified EBEs disclosed herein to other plants, cultivars and species and is useful for producing cultivars and novel types with the Xoo resistance trait.
The invention also provides methods for introducing the modified EBEs of the present invention into plants by crossing a plant which lacks the modified EBE with a plant that has the modified EBE, selfing the resulting generations and then selecting the plants exhibiting bacterial blight tolerance.
In another aspect, the invention provides a method for producing a hybrid seed comprising crossing a first plant parent with a second plant parent and harvesting the resultant hybrid seed, wherein either one or both parents contain modified EBEs. The hybrid seeds, plant and parts thereof produced by such method are also part of the invention.
In another aspect, the present invention provides for further single gene converted plants with modified EBEs. The desired further single transferred gene may preferably be a dominant or recessive allele. Preferably, the further single transferred gene will confer such traits as herbicide resistance, insect resistance, resistance for bacterial, fungal, or viral disease, male fertility, male sterility, enhanced nutritional quality, and industrial usage. The single gene may be a naturally occurring gene or a transgene introduced through genetic engineering techniques.
In another aspect, the present invention provides regenerable cells for use in tissue culture of a plant containing a modified EBE. The tissue culture will preferably be capable of regenerating plants having the physiological and morphological characteristics of the foregoing plant, and of regenerating plants having substantially the same genotype as the foregoing plant. Preferably, the regenerable cells in such tissue cultures will be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, pistils, roots, root tips, flowers, seeds, panicles or stems. Still further, the present invention provides plants regenerated from the tissue cultures of the invention.
The present invention also relates to a method of making a bacterial blight resistant plant comprising introducing to said plant a mutation in the EBE region of a bacterial susceptibility gene of said plant. This can be accomplished by any methodology for generating mutations including site directed mutagenesis, optimized directed evolution, Gene Site-Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR), error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, synthetic ligation reassembly (SLR) recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation, introduction of a TALEN fusion protein, or other targeted mutagenesis techniques.
In one preferred embodiment, site directed mutagenesis is accomplished by use of TALENs. This methodology is disclosed in United States Published Application US 2011-0201118 A1 which is hereby incorporated by reference in its entirety, particularly page 50, line 14-page 58, line 4 and FIGS. 1-3 and page 64, line 2-page 66, line 29 and FIGS. 10-16. Applicants have generated a TAL nuclease, a hybrid protein derived from FokI and AvrXa7, a member of transcription activator-like (TAL) effector family from phytopathogenic bacteria. The hybrid protein, referred to as TALEN, retains both recognition specificity for the EBE and the double-stranded DNA cleaving activity of FokI. The TALEN cleaves DNA adjacent to the AvrXa7-binding site under optimal conditions in vitro and when expressed promotes the DNA homologous recombination or paired target sequences.
Thus the invention comprises a method of introducing bacterial blight resistance to a cell by modifying the genetic material of a cell. The method includes providing a primary cell containing a chromosomal target DNA sequence (EBE) in which it is desired to have homologous recombination occur; providing a TAL effector endonuclease comprising an endonuclease domain that can cleave double stranded DNA, and a TAL effector domain comprising a plurality of TAL effector repeat sequences that, in combination, bind to a specific nucleotide sequence within the target DNA in the cell; and contacting the target DNA sequence with the TAL effector endonuclease in the cell such that the TAL effector endonuclease cleaves both strands of a nucleotide sequence within or adjacent to the target DNA sequence in the cell. The method can further include providing a nucleic acid comprising a sequence homologous to at least a portion of the target DNA, such that homologous recombination occurs between the target DNA sequence and the nucleic acid. The target DNA sequence is an endogenous EBE sequence that is endogenous to the cell and which provides for inhibition of induction, yet normal plant development. The contacting can include transfecting the cell with a vector comprising a TAL effector endonuclease coding sequence, and expressing the TAL effector endonuclease protein in the cell, mechanically injecting a TAL effector endonuclease protein into the cell, delivering a TAL effector endonuclease protein into the cell by means of the bacterial type III secretion system, or introducing a TAL effector endonuclease protein into the cell by electroporation. The endonuclease domain can be from a type II restriction endonuclease (e.g., FokI). The TAL effector domain that binds to a specific nucleotide sequence within the target DNA can include 15 or more DNA binding repeats.
In another embodiment the invention includes a method for designing a sequence specific TAL effector endonuclease capable of cleaving endogenous EBE DNA at a specific location. The method includes identifying a first unique endogenous chromosomal nucleotide sequence adjacent to a second nucleotide sequence (EBE) at which it is desired to introduce a double-stranded cut; and designing a sequence specific TAL effector endonuclease comprising (a) a plurality of DNA binding repeat domains that, in combination, bind to the first unique endogenous chromosomal nucleotide sequence, and (b) an endonuclease that generates a double-stranded cut and hence a mutation in the EBE site at the second nucleotide sequence.
According to the invention, the fusion protein can be expressed in a cell, e.g., by delivering the fusion protein to the cell or by delivering a polynucleotide encoding the fusion protein to a cell, wherein the polynucleotide, if DNA, is transcribed, and an RNA molecule delivered to the cell or a transcript of a DNA molecule delivered to the cell is translated, to generate the fusion protein. Methods for polynucleotide and polypeptide delivery to cells are known in the art and are presented elsewhere in this disclosure.
Targeted mutations in the EBE region resulting from the aforementioned method include, but are not limited to, point mutations (i.e., conversion of a single base pair to a different base pair), substitutions (i.e., conversion of a plurality of base pairs to a different sequence of identical length), insertions or one or more base pairs, deletions of one or more base pairs and any combination of the aforementioned sequence alterations.
The invention also includes a TAL effector endonuclease comprising an endonuclease domain and a TAL effector DNA binding domain specific for a particular DNA sequence. The TAL effector endonuclease (TALEN) can further include a purification tag. The endonuclease domain can be from a type II restriction endonuclease (e.g., FokI).
The invention also includes novel nucleic acid sequence encoding TALEN fusion proteins which have been designed to interact and cleave the target EBE sequences. In one embodiment the TALEN includes one or more of SEQ ID NOS 21 (dTALENR1) 22 (dTALENR2), and 23 (dTALENL1) operably linked to a promoter sequence to generate site directed nuclease fusions and mutant EBE recognition sites. In another embodiment, the TALEN includes one or more of SEQ ID NO:36 and 38 operably linked to a promoter sequence to generate site directed nuclease fusions and mutant EBE recognition sites. The method involves growing a host cell containing the nucleic acid molecules under conditions whereby the host cell expresses the same and nuclease fusion is conducted. Expression constructs comprising the nucleotide sequences encoding the fusion TALEN proteins operably linked to regulatory elements, vectors, and genetically modified plant cells expression the fusion proteins, and thus the resultant EBE modification are also included in the invention.
The invention further includes introducing the amino acid sequences of designed TALEN fusion proteins listed above as well as all conservatively modified variants, including SEQ ID NO:24, 25, 26, 37, and 39.
In another aspect of the invention, the invention comprises a method for producing a genetically modified plant that has improved tolerance to bacterial blight comprising the steps of: a) introducing into a plant seed, plant tissue or plant cell the TALEN expression construct as described above to produce a transformed plant seed, plant tissue or plant cell; and b) regenerating a transgenic plant from the transformed plant seed, transformed plant tissue or transformed plant cell, wherein the transgenic plant has improved tolerance to Xoo, or bacterial blight compared to a nonmodified plant. In one embodiment, the transgenic plant is a rice plant.
In another aspect of the invention, the invention comprises an immunoassay method to detect Xoo susceptibility in plants by screening the same for a TALEN fusion protein in said plant.
The invention further provides plants, seeds, and other plant parts such as pollen and ovules containing the modified EBE sequences, the TALEN fusion proteins, or the expression constructs encoding the TALEN fusion proteins disclosed herein.
In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by study of the following descriptions.



DESCRIPTION OF THE FIGURES
FIG. 1 demonstrates the high efficiency of targeted gene editing with TALENs. (A) Overlapping elements targeted by two pairs (Pair 1 and 2) of TALENs in the Os11N3 promoter. SEQ ID NO: 1 (Os11N3), SEQ ID NO: 16 (avrXa7 EBE) and SEQ ID NO: 17 (PthXo3 EBE) (B)-(D) Genotyping of T1 plants derived from the TALEN-expressed embryonic cells from independent transformation experiments (Exp.). Alleles containing wild type (wt), nucleotide insertion (+) and deletion (−) are separated by “/”. (E) Sequences of Os11N3 mutations induced by the Pair 1 nucleases with deletions (dashes) and insertions (red letters) (SEQ ID NOS 1-9 respectively). TALEN-binding sequences are underlined. (F) Os11N3 induction by AvrXa7 in plants of different genotypes. RT-PCR products are Os11N3 (gel a), Os04g19960 (gel b) and TFIIAγ5 (gel c) under treatments of Xoo strain ME2 (lane 1) and ME2 (avrXa7) (lane 2). (G) Resistance phenotype from one of T1 mutant plants.
FIG. 2 shows TALENs and their target DNA sequences in the promoter of chromosomal Os11N3 gene. Four basic modular repeats whose repeat variable diresidue (RVD) (colored blocks) recognizing one nucleotide (NT) in the target site are used to assemble the DNA binding domain of each designer TALEN. The two pairs of nucleases (Pair 1 and 2) are fusions between the DNA cleavage domain of Fold (FokI) and the native (AvrXa7) or customized TAL effector (dTALE). The last 40 amino acids at C-terminus of AvrXa7 and dTALE-L1 are truncated to avoid the inappropriate induction of Os11N3 by the activation domain (SEQ ID NO:1 and reverse complement). The other two TAL effectors (dTALE-R1 and dTALE-R2) contain the complete C-terminus. All four TAL effectors contain the complete N-terminus. Os11N3 promoter contains an effector binding element (EBE) for AvrXa7 (underlined in black), an EBE for PthXo3 (underlined in red) and the TATA box (boxed). Lower letters represent regions wherein two FokI domains dimerize and cause a double stranded DNA break.
FIG. 3 is a schematic of two gene expression cassettes in a single binary vector for rice transformation. The expression cassette includes a promoter [maize ubiquitin 1 promoter (Ubi1) and the cauliflower mosaic virus 35S gene promoter (35S)] (red arrow) and a terminator (Nos-T) (black bar) with the nuclease gene (TALEN-L and TALEN-R, respectively) (open box) inserted between.
FIG. 4 shows the sequences of Os11N3 mutations induced by the Pair 2 nucleases. Deletions and insertions are indicated by dashes and red letters, respectively. Number of nucleotide changes is indicated on the right side of sequence. TALEN-binding sequences are underlined. (SEQ ID NOS 1, 10, 11, 12, 13, 14, and 15 respectively)
FIG. 5 shows the TALENs for targeted editing of the promoter of Os8N3. A. The sucrose efflux transporter gene 11 (also called OsSWEET11) contains a PthXo1 binding site (shaded in grey). (A) pair of TALENs (SWT11-TALEN-L and -R) bind to the promoter of Os8N3 (underlined sequences with the L site starting at 86 bp upstream of the transcription initiation site). (SEQ ID NOS 36 and 38) (B) RVD sequences within the repeat domains of the paired TALENs and the corresponding EBEs. “*” represents the 13th amino acid missing in the last half repeat of the dTALENs. (SEQ ID NOS 37 and 39).
FIG. 6 shows how TALENs induce precise mutations at the PthXo1 binding site in the promoter of Os8N3 in rice. (A) Genotype of rice plant used for the second round of gene editing. The plants already contain a 4-bp deletion within the overlapping binding sites (shaded in grey, starting 32 bp upstream of the transcription initiation site of Os11N) by AvrXa7 and SPthXo3. (SEQ ID NOS 43 and 48) (B) Sequences of four representative OsN3 mutations induced by the TALENs with deletions (dashes) and insertion (red). TALEN binding sites are underlined and PthXo1 binding sequences are shaded in grey in wild type (wt). Number at the right side denotes the base pair deleted compared to wt. (SEQ ID NOS 44-48)
FIG. 7 shows disease resistance in transgenic rice T1 plants. (A) Resistance phenotype displayed by three T1 homozygous mutant plants compared to the susceptible phenotype of three plants homozygous for wild type Os8N3. The plants were inoculated with the pathogenic PXO99 that is dependent on PthXo1 to cause blight disease. (B) Lesion lengths caused by infection with the pathogenic PthXo1-dependent Xoo strain PXO99 strain were measured 14 days after inoculation of multiple plants (4-6 plants) homozygous for individual mutations or wild type segregated from individual mutant lines. The genotypes of the individually inoculated plants were confirmed by genotyping through sequencing. Leaf lesion lengths <5 cm indicate disease resistance and lesion lengths >10 indicate disease susceptibility. Error bars indicate 1 SD.
FIG. 8 shows sequences of the ORFs and the corresponding amino acid sequences of dTALENs (SEQ ID NOS:21-26).
FIG. 9 shows the sequences of the ORFs and the corresponding amino acid sequences of SWT11-TALEN-L and SWT11-TALEN-R (SEQ ID NOS:36-39).



DETAILED DESCRIPTION OF THE INVENTION
General
In order to provide a clear and consistent understanding of the specification and the claims, including the scope given to such terms, the following definitions are provided. Units, prefixes, and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. Unless otherwise provided for, software, electrical, and electronics terms as used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5th edition, 1993). The terms defined below are more fully defined by reference to the specification as a whole.
Practice of the methods, as well as preparation and use of the compositions disclosed herein employ, unless otherwise indicated, conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA and related fields as are within the skill of the art. These techniques are fully explained in the literature. See, for example, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) Humana Press, Totowa, 1999.
Definitions
The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer. The terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones). In general, an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of a corresponding naturally-occurring amino acids.
The term “introduced” in the context of inserting a nucleic acid into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNABinding” refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. Such interactions are generally characterized by a dissociation constant (Kd) of 10−6 M−1 or lower. “Affinity” refers to the strength of binding: increased binding affinity being correlated with a lower Kd.
As used herein “promoter” includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such as Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters which initiate transcription only in certain tissue are referred to as “tissue specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “repressible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most environmental conditions.
As used herein “recombinant” includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all as a result of deliberate human intervention. The term “recombinant” as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
As used herein, “vector” includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide. Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.
A “binding protein” is a protein that is able to bind non-covalently to another molecule. A binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein) and/or a protein molecule (a protein-binding protein). In the case of a protein-binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more molecules of a different protein or proteins. A binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding and protein-binding activity.
A “TAL effector DNA binding protein” (or binding domain) or a “TAL effector DNA recognition sequence” is a protein encompassing a series of repeat variable-diresidues (RVDs) within a larger protein, that binds DNA in a sequence-specific manner. The RVD regions of TAL effectors are polymorphisms within TALs typically at positions 12 and 13 in repeating units of typically 34 amino acids that bind for specific nucleotides and together with a plurality of repeating unit intervals make up the specific TAL effector DNA binding domain.
TAL effector DNA binding protein domains (their RVDs) can be “engineered” to bind to a predetermined nucleotide sequence. Non-limiting examples of methods for engineering the same are design and selection. A designed TAL effector DNA binding protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing RVD designs and binding data.
The term “sequence” refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double stranded. The term “donor sequence” refers to a nucleotide sequence that is inserted into a genome. A donor sequence can be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value there between or there above), preferably between about 100 and 1,000 nucleotides in length (or any integer there between), more preferably between about 200 and 500 nucleotides in length.
A “homologous, non-identical sequence” refers to a first sequence which shares a degree of sequence identity with a second sequence, but whose sequence is not identical to that of the second sequence. For example, a polynucleotide comprising the wild-type sequence of a mutant gene is homologous and non-identical to the sequence of the mutant gene. In certain embodiments, the degree of homology between the two sequences is sufficient to allow homologous recombination there between, utilizing normal cellular mechanisms. Two homologous non-identical sequences can be any length and their degree of non-homology can be as small as a single nucleotide (e.g., for correction of a genomic point mutation by targeted homologous recombination) or as large as 10 or more kilobases (e.g., for insertion of a gene at a predetermined ectopic site in a chromosome). Two polynucleotides comprising the homologous non-identical sequences need not be the same length. For example, an exogenous polynucleotide (i.e., donor polynucleotide) of between 20 and 10,000 nucleotides or nucleotide pairs can be used.
Techniques for determining nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this fashion. In general, identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity. The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the “BestFit” utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.). A preferred method of establishing percent identity in the context of the present disclosure is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects sequence identity. Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found at the following internet address: www.ncbi.nlm.gov. With respect to sequences described herein, the range of desired degrees of sequence identity is approximately 80% to 100% and any integer value therebetween. Typically the percent identities between sequences are at least 70-75%, preferably 80-82%, more preferably 85-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity.
Alternatively, the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that allow formation of stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. Two nucleic acid, or two polypeptide sequences are substantially homologous to each other when the sequences exhibit at least about 70%-75%, preferably 80%-82%, more preferably 85%-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity over a defined length of the molecules, as determined using the methods above. As used herein, substantially homologous also refers to sequences showing complete identity to a specified DNA or polypeptide sequence. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).
Selective hybridization of two nucleic acid fragments can be determined as follows. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit the hybridization of a completely identical sequence to a target molecule Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern (DNA) blot, Northern (RNA) blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a reference nucleic acid sequence, and then by selection of appropriate conditions the probe and the reference sequence selectively hybridize, or bind, to each other to form a duplex molecule. A nucleic acid molecule that is capable of hybridizing selectively to a reference sequence under moderately stringent hybridization conditions typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe. Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe. Hybridization conditions useful for probe/reference sequence hybridization, where the probe and reference sequence have a specific degree of sequence identity, can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).
Conditions for hybridization are well-known to those of skill in the art. Hybridization stringency refers to the degree to which hybridization conditions disfavor the formation of hybrids containing mismatched nucleotides, with higher stringency correlated with a lower tolerance for mismatched hybrids. Factors that affect the stringency of hybridization are well-known to those of skill in the art and include, but are not limited to, temperature, pH, ionic strength, and concentration of organic solvents such as, for example, formamide and dimethylsulfoxide. As is known to those of skill in the art, hybridization stringency is increased by higher temperatures, lower ionic strength and lower solvent concentrations.
With respect to stringency conditions for hybridization, it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of the sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions. The selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
“Recombination” refers to a process of exchange of genetic information between two polynucleotides. For the purposes of this disclosure, “homologous recombination (HR)” refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells. This process requires nucleotide sequence homology, uses a “donor” molecule to template repair of a “target” molecule (i.e., the one that experienced the double-strand break), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or “synthesis-dependent strand annealing,” in which the donor is used to resynthesize genetic information that will become part of the target, and/or related processes. Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.
“Cleavage” refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, fusion polypeptides are used for targeted double-stranded DNA cleavage.
A “cleavage domain” comprises one or more polypeptide sequences which possesses catalytic activity for DNA cleavage. A cleavage domain can be contained in a single polypeptide chain or cleavage activity can result from the association of two (or more) polypeptides.
“Chromatin” is the nucleoprotein structure comprising the cellular genome. Cellular chromatin comprises nucleic acid, primarily DNA, and protein, including histones and non-histone chromosomal proteins. The majority of eukaryotic cellular chromatin exists in the form of nucleosomes, wherein a nucleosome core comprises approximately 150 base pairs of DNA associated with an octamer comprising two each of histones H2A, H2B, H3 and H4; and linker DNA (of variable length depending on the organism) extends between nucleosome cores. A molecule of histone H1 is generally associated with the linker DNA. For the purposes of the present disclosure, the term “chromatin” is meant to encompass all types of cellular nucleoprotein, both prokaryotic and eukaryotic. Cellular chromatin includes both chromosomal and episomal chromatin.
A “chromosome,” is a chromatin complex comprising all or a portion of the genome of a cell. The genome of a cell is often characterized by its karyotype, which is the collection of all the chromosomes that comprise the genome of the cell. The genome of a cell can comprise one or more chromosomes.
An “accessible region” is a site in cellular chromatin in which a target site present in the nucleic acid can be bound by an exogenous molecule which recognizes the target site. Without wishing to be bound by any particular theory, it is believed that an accessible region is one that is not packaged into a nucleosomal structure. The distinct structure of an accessible region can often be detected by its sensitivity to chemical and enzymatic probes, for example, nucleases.
A “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist. For example, the sequence 5′-GAATTC-3′ is a target site for the Eco RI restriction endonuclease.
A “heterologous” molecule is a molecule that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods. “Normal presence in the cell” is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of muscle is an exogenous molecule with respect to an adult muscle cell. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to a non-heat-shocked cell. An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally-functioning endogenous molecule.
A heterologous molecule can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules. Nucleic acids include DNA and RNA, can be single- or double-stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Pat. Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases and helicases.
A heterologous molecule can be the same type of molecule as an endogenous molecule, e.g., a heterologous protein or nucleic acid. For example, a heterologous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell. Methods for the introduction of exogenous molecules into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer.
By contrast, an “endogenous” molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally-occurring episomal nucleic acid. Additional endogenous molecules can include proteins, for example, transcription factors and enzymes.
A “fusion” molecule is a molecule in which two or more subunit molecules are linked, preferably covalently. The subunit molecules can be the same chemical type of molecule, or can be different chemical types of molecules. Examples of the first type of fusion molecule include, but are not limited to, fusion proteins (for example, a fusion between a TAL effector sequence DNA-binding domain and a cleavage domain) and fusion nucleic acids (for example, a nucleic acid encoding the fusion protein described supra). Examples of the second type of fusion molecule include, but are not limited to, a fusion between a triplex-forming nucleic acid and a polypeptide, and a fusion between a minor groove binder and a nucleic acid.
Expression of a fusion protein in a cell can result from delivery of the fusion protein to the cell or by delivery of a polynucleotide encoding the fusion protein to a cell, wherein the polynucleotide is transcribed, and the transcript is translated, to generate the fusion protein. Trans-splicing, polypeptide cleavage and polypeptide ligation can also be involved in expression of a protein in a cell. Methods for polynucleotide and polypeptide delivery to cells are presented elsewhere in this disclosure.
A “gene,” for the purposes of the present disclosure, includes a DNA region encoding a gene product (see infra), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.
“Gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
“Modulation” of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression.
A “region of interest” is any region of cellular chromatin, such as, for example, a gene or a non-coding sequence within or adjacent to a gene, in which it is desirable to bind an exogenous molecule. Binding can be for the purposes of targeted DNA cleavage and/or targeted recombination. A region of interest can be present in a chromosome, an episome, an organellar genome (e.g., mitochondrial, chloroplast), or an infecting viral genome, for example. A region of interest can be within the coding region of a gene, within transcribed non-coding regions such as, for example, leader sequences, trailer sequences or introns, or within non-transcribed regions, either upstream or downstream of the coding region. A region of interest can be as small as a single nucleotide pair or up to 2,000 nucleotide pairs in length, or any integral value of nucleotide pairs.
The terms “operative linkage” and “operatively linked” (or “operably linked”) are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. By way of illustration, a transcriptional regulatory sequence, such as a promoter, is operatively linked to a coding sequence if the transcriptional regulatory sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptional regulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence, even though they are not contiguous.
A “functional fragment” of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains the same function as the full-length protein, polypeptide or nucleic acid. A functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well-known in the art. Similarly, methods for determining protein function are well-known. For example, the DNA-binding function of a polypeptide can be determined, for example, by filter-binding, electrophoretic mobility-shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. See Ausubel et al., supra. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays or complementation, both genetic and biochemical. See, for example, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No. 5,585,245 and PCT WO 98/44350.
The invention includes plants with plant susceptibility genes that have been modified in the EBE region. This can include modified Os11N3 EBE nucleic acid sequences SEQ ID NOS: 5, 6, 7, 9, 44, 45, 46, or 47 which provide resistance to Xoo-mediated induction and bacterial blight, but which also maintain normal plant growth and development. In another embodiment, a nucleotide sequence that is at least 90 or greater, 95% or greater percent similar to SEQ ID NO: 5, 6, 7, or 9 which still inhibits AvrXa7 or PthXo3 induction yet retains normal plant development. In another embodiment, a nucleotide sequence that is at least 90 or greater, 95% or greater percent similar to SEQ ID NO: 44, 45, 46, or 47 which still inhibits PthXo1 induction yet retains normal plant development. In yet another embodiment, a nucleotide sequence that hybridizes to SEQ ID NO 5, 6, 7, or 9 under stringent hybridization conditions involving, for example, hybridization in a hybridization buffer containing, for example, 20 percent formamide in 0.9M saline/0.09M SSC buffer, at a temperature of about 42° C. which still inhibits AvrXa7 or PthXo3 induction yet retains normal plant development are within the scope of the invention. In yet another embodiment, a nucleotide sequence that hybridizes to SEQ ID NO 44, 45, 46, or 47 under stringent hybridization conditions involving, for example, hybridization in a hybridization buffer containing, for example, 20 percent formamide in 0.9M saline/0.09M SSC buffer, at a temperature of about 42° C. which still inhibits PthXo1 induction yet retains normal plant development are also within the scope of the invention.
Bacterial blight resistance plants may be obtained by incorporating the modified EBE regions of the invention into a plant. This may be accomplished by back crossing and standard breeding techniques or by site directed mutagenesis techniques. One particularly preferred site directed mutagenesis technique is via TALE nucleases (TALENs), the hybrid proteins of native or customized TAL effectors and the DNA cleavage domains such as FokI, using TALENs to mutate S gene EBEs in a way the renders it no longer responsive to the cognate TAL effector and, thus, confers resistance to bacterial blight.
EBE Target Sites
The disclosed methods and compositions of the invention include specific target sites on EBE regions that may be modified according to the invention to impart disease resistance while still retaining plant growth and function. In the TAL effector site directed mutagenesis embodiment, the invention includes fusion proteins such as SEQ ID NOS 24, 25, 26, 37 or 39 comprising a cleavage domain and a TAL effector DNA binding domain, or DNA recognition sequence in which the RVDs, by binding to a sequence in cellular chromatin (e.g., a target site or a binding site), directs the activity of the cleavage domain (or cleavage half-domain) to the vicinity of the sequence and, hence, induces cleavage in the vicinity of the target sequence, here the EBE site. As set forth elsewhere in this disclosure, Particular RVDs within a TAL binding domain can be engineered to bind to virtually any desired sequence and here are engineered to bind to EBE sites. Accordingly, after identifying a region of interest containing a sequence at which cleavage or recombination is desired, one or more TAL effector DNA binding domains can be engineered to bind to one or more sequences in the region of interest. Expression of a fusion protein comprising a TAL effector DNA binding domain and a cleavage domain, in a cell, effects cleavage in the region of interest.
Selection of a sequence in cellular chromatin for binding by a TAL effector binding domain (e.g., a target site) may be determined by identifying EBE sites within susceptibility genes for bacterial pathogens that are upregulated or otherwise modified by bacterial pathogen infection.
Sequence-Specific Endonucleases
Sequence-specific nucleases such as SEQ ID NOS 24, 25, 26, 37 and 39 and recombinant nucleic acids encoding the sequence-specific endonucleases SEQ ID NOS: 21, 22, 23, 36 and 38 are provided herein. The sequence-specific endonucleases can include TAL effector DNA binding domains and endonuclease domains. Thus, nucleic acids encoding such sequence-specific endonucleases can include a nucleotide sequence from a sequence-specific TAL effector linked to a nucleotide sequence from a nuclease.
TAL effectors are proteins of plant pathogenic bacteria that are injected by the pathogen into the plant cell, where they travel to the nucleus and function as transcription factors to turn on specific plant genes. The primary amino acid sequence of a TAL effector dictates the nucleotide sequence to which it binds. Because the relationship between the TAL amino acid sequence and the target binding site is simple, target sites can be predicted for TAL effectors, and TAL effectors also can be engineered and generated for the purpose of binding to particular nucleotide sequences.
Fused to the TAL effector-encoding nucleic acid sequences are sequences encoding a nuclease or a portion of a nuclease, typically a nonspecific cleavage domain from a type II restriction endonuclease such as FokI (Kim et al. (1996) Proc. Natl. Acad Sci. USA 93:1156-1160). Other useful endonucleases may include, for example, HhaI, HindIII, NotI, BbvC1, EcoRI, BglI, and AlwI. The fact that some endonucleases (e.g., FokI) only function as dimers can be capitalized upon to enhance the target specificity of the TAL effector. For example, in some cases each FokI monomer can be fused to a TAL effector sequence that recognizes a different DNA target sequence, and only when the two recognition sites are in close proximity do the inactive monomers come together to create a functional enzyme. By requiring DNA binding to activate the nuclease, a highly site-specific restriction enzyme can be created.
A sequence-specific TAL effector endonuclease as provided herein can recognize a particular sequence within a preselected target nucleotide sequence present in a cell. Thus, in some embodiments, a target nucleotide sequence can be scanned for nuclease recognition sites, and a particular nuclease can be selected based on the target sequence. In other cases, a TAL effector endonuclease can be engineered to target a particular cellular sequence. A nucleotide sequence encoding the desired TAL effector endonuclease can be inserted into any suitable expression vector, and can be linked to one or more expression control sequences. For example, a nuclease coding sequence can be operably linked to a promoter sequence that will lead to constitutive expression of the endonuclease in the species of plant to be transformed. Alternatively, an endonuclease coding sequence can be operably linked to a promoter sequence that will lead to conditional expression (e.g., expression under certain nutritional conditions).
Cleavage Domains
The cleavage domain portion of the fusion proteins disclosed herein can be obtained from any endo- or exonuclease. Exemplary endonucleases from which a cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes which cleave DNA are known (e.g., S1 Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains.
Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding. Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. For example, the Type IIS enzyme FokI catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise the cleavage domain (or cleavage half-domain) from at least one Type IIS restriction enzyme.
An exemplary Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is FokI. This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575. Accordingly, for the purposes of the present disclosure, the portion of the FokI enzyme used in the disclosed fusion proteins is considered a cleavage half-domain. Thus, for targeted double-stranded cleavage and/or targeted replacement of cellular sequences using TAL-FokI fusions, two fusion proteins, each comprising a FokI cleavage half-domain, can be used to reconstitute a catalytically active cleavage domain. Parameters for targeted cleavage and targeted sequence alteration using TAL-FokI fusions are provided elsewhere in this disclosure.
A cleavage domain or cleavage half-domain can be any portion of a protein that retains cleavage activity, or that retains the ability to multimerize (e.g., dimerize) to form a functional cleavage domain.
Additional restriction enzymes also contain separable binding and cleavage domains, and these are contemplated by the present disclosure. See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420. Examples of Type IIS Restriction Enzymes include: Aar I, BsrB I, SspD5 I, Ace III, BsrD I, Sth132 I, Aci I, BstF5 I, Sts I, Alo I, Btr I, TspDT I, Bae I, Bts I, TspGW I, Bbr7 I, Cdi I, Tth111 II, Bbv I, CjeP I, UbaP I, Bbv II, Drd II, Bsa I, BbvC I, Eci I, BsmB I, Bcc I, Eco3I, Bce83 I, Eco57 I, BceA I, Eco57M I, Bcef I, Esp3 I, Bcg I, Fau I, BciV I, Fin I, Bfi I, Fok I, Bin I, Gdi II, Bmg I, Gsu I, Bpu10 I, Hga I, BsaX I, Hin4 II, Bsb I, Hph I, BscA I, Ksp632 I, BscG I, Mbo II, BseR I, Mly I, BseY I, Mme I, Bsi I, Mnl I, Bsm I, Pfl1108 I, BsmA I, Ple I, BsmF I, Ppi I, Bsp24 I, Psr I, BspG I, RleA I, BspM I, Sap I, BspNC I, SfaN I, Bsr I, and Sim I
TAL Effector DNA Domain-Cleavage Domain Fusions
Methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art. For example, methods for the design and construction of fusion protein comprising TAL proteins (and polynucleotides encoding same) are described in U.S. Pat. Nos. 6,453,242 and 6,534,261. In certain embodiments, polynucleotides encoding such fusion proteins are constructed. These polynucleotides can be inserted into a vector and the vector can be introduced into a cell (see below for additional disclosure regarding vectors and methods for introducing polynucleotides into cells).
In certain embodiments of the methods described herein, a fusion protein comprises the overlapping a TAL effector binding domain from AvrXa7 and PthXo3 (SEQ ID NO:20) and a cleavage half-domain from the FokI restriction enzyme, and two such fusion proteins are expressed in a cell. Expression of two fusion proteins in a cell can result from delivery of the two proteins to the cell; delivery of one protein and one nucleic acid encoding one of the proteins to the cell; delivery of two nucleic acids, each encoding one of the proteins, to the cell; or by delivery of a single nucleic acid, encoding both proteins, to the cell. In additional embodiments, a fusion protein comprises a single polypeptide chain comprising two cleavage half domains and the overlapping TAL AvrXa7 and PthXo3 (SEQ ID NO:20) binding domain. In this case, a single fusion protein is expressed in a cell and, without wishing to be bound by theory, is believed to cleave DNA as a result of formation of an intramolecular dimer of the cleavage half-domains.
In certain embodiments, the components of the fusion proteins (e.g, TAL-FokI fusions) are arranged such that the cleavage domain is nearest the amino terminus of the fusion protein, and the TAL domain is nearest the carboxy-terminus. This provides certain advantages such as the retention of the transcription activator activity which enables one to measure the DNA binding specificity of naturally occurring TAL or newly engineered TAL used for nuclease fusion and this orientation may give the flexibility of spacer lengths.
Methods for Targeted Cleavage
The disclosed methods and compositions can be used to cleave DNA at an EBE in cellular chromatin (e.g., at a desired or predetermined site in a genome, for example, in a gene, either mutant or wild-type). For such targeted DNA cleavage, TAL binding domain is engineered to bind a target site at or near the predetermined EBE cleavage site, and a fusion protein comprising the engineered TAL binding domain and a cleavage domain is expressed in a cell. Upon binding of the TAL RVDs portion of the fusion protein to the target site, the DNA is cleaved near the target site by the cleavage domain.
For targeted cleavage using a TAL binding domain-cleavage domain fusion polypeptide, the binding site can encompass the cleavage site, or the near edge of the binding site can be 1, 2, 3, 4, 5, 6, 10, 25, 50 or more nucleotides (or any integral value between 1 and 50 nucleotides) from the cleavage site. The exact location of the binding site, with respect to the cleavage site, will depend upon the particular cleavage domain, and the length of any linker.
Thus, the methods described herein can employ an engineered TAL effector DNA binding domain fused to a cleavage domain. In these cases, the binding domain is engineered to bind to a target sequence, at or near which cleavage is desired. The fusion protein, or a polynucleotide encoding same, is introduced into a cell. Once introduced into, or expressed in, the cell, the fusion protein binds to the target sequence and cleaves at or near the target sequence. The exact site of cleavage depends on the nature of the cleavage domain and/or the presence and/or nature of linker sequences between the binding and cleavage domains. Optimal levels of cleavage can also depend on both the distance between the binding sites of the two fusion proteins (See, for example, Smith et al. (2000) Nucleic Acids Res. 28:3361-3369; Bibikova et al. (2001) Mol. Cell. Biol. 21:289-297) and the length of the ZC linker in each fusion protein.
In certain embodiments, the cleavage domain comprises two cleavage half-domains, both of which are part of a single polypeptide comprising a binding domain, a first cleavage half-domain and a second cleavage half-domain. The cleavage half-domains can have the same amino acid sequence or different amino acid sequences, so long as they function to cleave the DNA.
Cleavage half-domains may also be provided in separate molecules. For example, two fusion polypeptides may be introduced into a cell, wherein each polypeptide comprises a binding domain and a cleavage half-domain. The cleavage half-domains can have the same amino acid sequence or different amino acid sequences, so long as they function to cleave the DNA. Further, the binding domains bind to target sequences which are typically disposed in such a way that, upon binding of the fusion polypeptides, the two cleavage half-domains are presented in a spatial orientation to each other that allows reconstitution of a cleavage domain (e.g., by dimerization of the half-domains), thereby positioning the half-domains relative to each other to form a functional cleavage domain, resulting in cleavage of cellular chromatin in a region of interest. Generally, cleavage by the reconstituted cleavage domain occurs at a site located between the two target sequences. One or both of the proteins can be engineered to bind to its target site.
The two fusion proteins can bind in the region of interest in the same or opposite polarity, and their binding sites (i.e., target sites) can be separated by any number of nucleotides, e.g., from 0 to 200 nucleotides or any integral value therebetween. In certain embodiments, the binding sites for two fusion proteins, each comprising a TAL effector binding domain and a cleavage half-domain, can be located between 5 and 18 nucleotides apart, for example, 5-8 nucleotides apart, or 15-18 nucleotides apart, or 6 nucleotides apart, or 16 nucleotides apart, as measured from the edge of each binding site nearest the other binding site, and cleavage occurs between the binding sites.
The site at which the DNA is cleaved generally lies between the binding sites for the two fusion proteins. Double-strand breakage of DNA often results from two single-strand breaks, or “nicks,” offset by 1, 2, 3, 4, 5, 6 or more nucleotides, (for example, cleavage of double-stranded DNA by native FokI results from single-strand breaks offset by 4 nucleotides). Thus, cleavage does not necessarily occur at exactly opposite sites on each DNA strand. In addition, the structure of the fusion proteins and the distance between the target sites can influence whether cleavage occurs adjacent a single nucleotide pair, or whether cleavage occurs at several sites. However, for many applications, including targeted recombination and targeted mutagenesis (see infra) cleavage within a range of nucleotides is generally sufficient, and cleavage between particular base pairs is not required.
As noted above, the fusion protein(s) can be introduced as polypeptides and/or polynucleotides. For example, two polynucleotides, each comprising sequences encoding one of the aforementioned polypeptides, can be introduced into a cell, and when the polypeptides are expressed and each binds to its target sequence, cleavage occurs at or near the target sequence. Alternatively, a single polynucleotide comprising sequences encoding both fusion polypeptides is introduced into a cell. Polynucleotides can be DNA, RNA or any modified forms or analogues or DNA and/or RNA.
In addition to the fusion molecules described herein, targeted replacement of a selected genomic sequence also requires the introduction of the replacement (or donor) sequence. The donor sequence can be introduced into the cell prior to, concurrently with, or subsequent to, expression of the fusion protein(s). The donor polynucleotide contains sufficient homology to a genomic sequence to support homologous recombination between it and the genomic sequence to which it bears homology. Approximately 25, 50, 100 or 200 nucleotides or more of sequence homology between a donor and a genomic sequence (or any integral value between 10 and 200 nucleotides, or more) will support homologous recombination therebetween. Donor sequences can range in length from 10 to 5,000 nucleotides (or any integral value of nucleotides therebetween) or longer. It will be readily apparent that the donor sequence is typically not identical to the genomic sequence that it replaces. For example, the sequence of the donor polynucleotide can contain one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, so long as sufficient homology is present to support homologous recombination. Alternatively, a donor sequence can contain a non-homologous sequence flanked by two regions of homology. Additionally, donor sequences can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin. Generally, the homologous region(s) of a donor sequence will have at least 50% sequence identity to a genomic sequence with which recombination is desired. In certain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity can be present, depending upon the length of the donor polynucleotide.
A donor molecule can contain several, discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of sequences not normally present in a region of interest, said sequences can be present in a donor nucleic acid molecule and flanked by regions of homology to sequence in the region of interest.
To simplify assays (e.g., hybridization, PCR, restriction enzyme digestion) for determining successful insertion of the donor sequence, certain sequence differences may be present in the donor sequence as compared to the genomic sequence. Preferably, if located in a coding region, such nucleotide sequence differences will not change the amino acid sequence, or will make silent amino acid changes (i.e., changes which do not affect the structure or function of the protein). The donor polynucleotide can optionally contain changes in sequences corresponding to the TAL effector domain binding (or recognition) sites in the region of interest, to prevent cleavage of donor sequences that have been introduced into cellular chromatin by homologous recombination.
The donor polynucleotide can be DNA or RNA, single-stranded or double-stranded and can be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3′ terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. A polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV).
Without being bound by one theory, it appears that the presence of a double-stranded break in a cellular sequence, coupled with the presence of an exogenous DNA molecule having homology to a region adjacent to or surrounding the break, activates cellular mechanisms which repair the break by transfer of sequence information from the donor molecule into the cellular (e.g., genomic or chromosomal) sequence; i.e., by a processes of homologous recombination. Applicants' methods advantageously combine the powerful targeting capabilities of engineered TALs with a cleavage domain (or cleavage half-domain) to specifically target a double-stranded break to the region of the genome at which recombination is desired.
For alteration of a chromosomal sequence, it is not necessary for the entire sequence of the donor to be copied into the chromosome, as long as enough of the donor sequence is copied to effect the desired sequence alteration.
Expression Vectors
A nucleic acid encoding one or more fusion proteins can be cloned into a vector for transformation into prokaryotic or eukaryotic cells for replication and/or expression. Vectors can be prokaryotic vectors, e.g., plasmids, or shuttle vectors, insect vectors, or eukaryotic vectors. A nucleic acid encoding a TAL effector binding domain can also be cloned into an expression vector, for administration to a plant cell, animal cell, preferably a mammalian cell or a human cell, fungal cell, bacterial cell, or protozoal cell.
To obtain expression of a cloned gene or nucleic acid, sequences encoding a fusion protein are typically subcloned into an expression vector that contains a promoter to direct transcription.
Promoters are involved in recognition and binding of RNA polymerase and other proteins to initiate and modulate transcription. To bring a coding sequence under the control of a promoter, it typically is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. A promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation start site, or about 2,000 nucleotides upstream of the transcription start site. A promoter typically comprises at least a core (basal) promoter. A promoter also may include at least one control element such as an upstream element. Such elements include upstream activation regions (UARs) and, optionally, other DNA sequences that affect transcription of a polynucleotide such as a synthetic upstream element.
The choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell or tissue specificity. For example, tissue-, organ- and cell-specific promoters that confer transcription only or predominantly in a particular tissue, organ, and cell type, respectively, can be used. In some embodiments, promoters specific to vegetative tissues such as the stem, parenchyma, ground meristem, vascular bundle, cambium, phloem, cortex, shoot apical meristem, lateral shoot meristem, root apical meristem, lateral root meristem, leaf primordium, leaf mesophyll, or leaf epidermis can be suitable regulatory regions. In some embodiments, promoters that are essentially specific to seeds (“seed-preferential promoters”) can be useful. Seed-specific promoters can promote transcription of an operably linked nucleic acid in endosperm and cotyledon tissue during seed development. Alternatively, constitutive promoters can promote transcription of an operably linked nucleic acid in most or all tissues of a plant, throughout plant development. Other classes of promoters include, but are not limited to, inducible promoters, such as promoters that confer transcription in response to external stimuli such as chemical agents, developmental stimuli, or environmental stimuli.
A basal promoter is the minimal sequence necessary for assembly of a transcription complex required for transcription initiation. Basal promoters frequently include a “TATA box” element that may be located between about 15 and about 35 nucleotides upstream from the site of transcription initiation. Basal promoters also may include a “CCAAT box” element (typically the sequence CCAAT) and/or a GGGCG sequence, which can be located between about 40 and about 200 nucleotides, typically about 60 to about 120 nucleotides, upstream from the transcription start site.
Non-limiting examples of promoters that can be included in the nucleic acid constructs provided herein include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1′ or 2′ promoters derived from T-DNA of Agrobacterium tumefaciens, promoters from a maize leaf-specific gene described by Busk ((1997) Plant J 11:1285-1295), kn1-related genes from maize and other species, and transcription initiation regions from various plant genes such as the maize ubiquitin-1 promoter.
A 5′ untranslated region (UTR) is transcribed, but is not translated, and lies between the start site of the transcript and the translation initiation codon and may include the +1 nucleotide. A 3′ UTR can be positioned between the translation termination codon and the end of the transcript. UTRs can have particular functions such as increasing mRNA message stability or translation attenuation. Examples of 3′ UTRs include, but are not limited to polyadenylation signals and transcription termination sequences. A polyadenylation region at the 3′-end of a coding region can also be operably linked to a coding sequence. The polyadenylation region can be derived from the natural gene, from various other plant genes, or from an Agrobacterium T-DNA.
The vectors provided herein also can include, for example, origins of replication, and/or scaffold attachment regions (SARs). In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag” tag (Kodak, New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.
It will be understood that more than one regulatory region may be present in a recombinant polynucleotide, e.g., introns, enhancers, upstream activation regions, and inducible elements.
Recombinant nucleic acid constructs can include a polynucleotide sequence inserted into a vector suitable for transformation of cells (e.g., plant cells or animal cells). Recombinant vectors can be made using, for example, standard recombinant DNA techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989; 3rd ed., 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., supra. Bacterial expression systems for expressing the ZFP are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983)). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known by those of skill in the art and are also commercially available.
The promoter used to direct expression of a TAL-cleavage domain fusion protein-encoding nucleic acid depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification of TAL-cleavage domain fusion proteins. In contrast, when a TAL-cleavage domain fusion protein is administered in vivo for gene regulation, either a constitutive or an inducible promoter is used, depending on the particular use of the TAL-cleavage domain fusion protein. In addition, a preferred promoter for administration of a TAL-cleavage domain fusion protein can be a weak promoter, such as HSV TK or a promoter having similar activity. The promoter typically can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tet-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, PNAS 89:5547 (1992); Oligino et al., Gene Ther. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441 (1997); Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al., Nat. Biotechnol. 16:757-761 (1998)). The MNDU3 promoter can also be used, and is preferentially active in CD34+ hematopoietic stem cells.
In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic. A typical expression cassette thus contains a promoter operably linked, e.g., to a nucleic acid sequence encoding the TAL-cleavage domain fusion protein and signals required, e.g., for efficient polyadenylation of the transcript, transcriptional termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous splicing signals.
The particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the TAL-cleavage domain fusion protein, e.g., expression in plants, animals, bacteria, fungus, protozoa, etc. (see expression vectors described below). Standard bacterial expression vectors include plasmids such as pBR322-based plasmids, pSKF, pET23D, and commercially available fusion expression systems such as GST and LacZ. An exemplary fusion protein is the maltose binding protein, “MBP.” Such fusion proteins are used for purification of the TAL-cleavage domain fusion protein. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, for monitoring expression, and for monitoring cellular and subcellular localization, e.g., c-myc or FLAG.
Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
Some expression systems have markers for selection of stably transfected cell lines such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. High yield expression systems are also suitable, such as using a baculovirus vector in insect cells, with a TAL-cleavage domain fusion protein encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of recombinant sequences.
Standard transfection methods are used to produce plant, bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).
Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, ultrasonic methods (e.g., sonoporation), liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the protein of choice.
Nucleic Acids Encoding Fusion Proteins and Delivery to Cells
Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding engineered TAL-cleavage domain fusion proteins in animal cells (e.g., mammalian cells) and target tissues. Such methods can also be used to administer nucleic acids encoding TAL-cleavage domain fusion proteins to cells in vitro. In certain embodiments, nucleic acids encoding TAL-cleavage domain fusion proteins are administered for in vivo or ex vivo gene therapy uses. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bohm (eds) (1995); and Yu et al., Gene Therapy 1: 13-26 (1994).
Methods of non-viral delivery of nucleic acids encoding engineered TAL-cleavage domain fusion proteins include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids.
Additional exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.) and BTX Molecular Delivery Systems (Holliston, Mass.).
The use of RNA or DNA viral based systems for the delivery of nucleic acids encoding engineered TAL-cleavage domain fusion proteins take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo). Conventional viral based systems for the delivery of TAL-cleavage domain fusion proteins include, but are not limited to, retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
In applications in which transient expression of a TAL-cleavage domain fusion protein fusion protein is preferred, adenoviral based systems can be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (“AAV”) vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).
Replication-deficient recombinant adenoviral vectors (Ad) can be produced at high titer and readily infect a number of different cell types. Most adenovirus vectors are engineered such that a transgene replaces the Ad E1a, E1b, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity. An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al., Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).
Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and .psi.2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
In many gene therapy applications, it is desirable that the gene therapy vector be delivered with a high degree of specificity to a particular tissue type. Accordingly, a viral vector can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus. The ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al., Proc. Natl. Acad. Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. This principle can be extended to other virus-target cell pairs, in which the target cell expresses a receptor and the virus expresses a fusion protein comprising a ligand for the cell-surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to nonviral vectors. Such vectors can be engineered to contain specific uptake sequences which favor uptake by specific target cells.
Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
Ex vivo cell transfection for diagnostics, research, or for gene therapy (e.g., via re-infusion of the transfected cells into the host organism) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from the subject organism, transfected with a ZFP nucleic acid (gene or cDNA), and re-infused back into the subject organism (e.g., patient). Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al., Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) and the references cited therein for a discussion of how to isolate and culture cells from patients).
In one embodiment, stem cells are used in ex vivo procedures for cell transfection and gene therapy. The advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow. Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN-γ and TNF-α are known (see Inaba et al., J. Exp. Med. 176:1693-1702 (1992)).
Stem cells are isolated for transduction and differentiation using known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1 (granulocytes), and lad (differentiated antigen presenting cells) (see Inaba et al., J. Exp. Med. 176:1693-1702 (1992)).
Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing therapeutic TAL-cleavage domain fusion protein nucleic acids can also be administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available, as described below (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).
With further respect to plants, the polynucleotides and vectors described herein can be used to transform a number of monocotyledonous and dicotyledonous plants and plant cell systems, including dicots such as safflower, alfalfa, soybean, coffee, amaranth, rapeseed (high erucic acid and canola), peanut or sunflower, as well as monocots such as oil palm, sugarcane, banana, sudangrass, com, wheat, rye, barley, oat, rice, millet, or sorghum. Also suitable are gymnosperms such as fir and pine.
Thus, the methods described herein can be utilized with dicotyledonous plants belonging, for example, to the orders Magniolales, Illiciales, Laurales, Piperales, Aristochiales, Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales, Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales, Lecythidales, Violates, Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales, Myrtales, Cornales, Proteales, San tales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales, Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales, and Asterales. The methods described herein also can be utilized with monocotyledonous plants such as those belonging to the orders Alismatales, Hydrocharitales, Najadales, Triuridales, Commelinales, Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales, Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales, Lilliales, and Orchid ales, or with plants belonging to Gymnospermae, e.g., Pinales, Ginkgoales, Cycadales and Gnetales.
The methods can be used over a broad range of plant species, including species from the dicot genera Atropa, Alseodaphne, Anacardium, Arachis, Beilschmiedia, Brassica, Carthamus, Cocculus, Croton, Cucumis, Citrus, Citrullus, Capsicum, Catharanthus, Cocos, Coffea, Cucurbita, Daucus, Duguetia, Eschscholzia, Ficus, Fragaria, Glaucium, Glycine, Gossypium, Helianthus, Hevea, Hyoscyamus, Lactuca, Landolphia, Linum, Litsea, Lycopersicon, Lupinus, Manihot, Majorana, Malus, Medicago, Nicotiana, Olea, Parthenium, Papaver, Persea, Phaseolus, Pistacia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Senecio, Sinomenium, Stephania, Sinapis, Solanum, Theobroma, Trifolium, Trigonella, Vicia, Vinca, Vilis, and Vigna; the monocot genera Allium, Andropogon, Aragrostis, Asparagus, Avena, Cynodon, Elaeis, Festuca, Festulolium, Heterocallis, Hordeum, Lemna, Lolium, Musa, Oryza, Panicum, Pannesetum, Phleum, Poa, Secale, Sorghum, Triticum, and Zea; or the gymnosperm genera Abies, Cunninghamia, Picea, Pinus, and Pseudotsuga. 
A transformed cell, callus, tissue, or plant can be identified and isolated by selecting or screening the engineered cells for particular traits or activities, e.g., those encoded by marker genes or antibiotic resistance genes. Such screening and selection methodologies are well known to those having ordinary skill in the art. In addition, physical and biochemical methods can be used to identify transformants. These include Southern analysis or PCR amplification for detection of a polynucleotide; Northern blots, S1 RNase protection, primer-extension, or RT-PCR amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides; and protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polypeptides and/or polynucleotides. Methods for performing all of the referenced techniques are well known. Polynucleotides that are stably incorporated into plant cells can be introduced into other plants using, for example, standard breeding techniques.
DNA constructs may be introduced into the genome of a desired plant host by a variety of conventional techniques. For reviews of such techniques see, for example, Weissbach & Weissbach Methods for Plant Molecular Biology (1988, Academic Press, N.Y.) Section VIII, pp. 421-463; and Grierson & Corey, Plant Molecular Biology (1988, 2d Ed.), Blackie, London, Ch. 7-9. For example, the DNA construct may 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 tissue using biolistic methods, such as DNA particle bombardment (see, e.g., Klein et al (1987) Nature 327:70-73). Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al (1984) Science 233:496-498, and Fraley et al (1983) Proc. Nat'l. Acad. Sci. USA 80:4803. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria using binary T DNA vector (Bevan (1984) Nuc. Acid Res. 12:8711-8721) or the co-cultivation procedure (Horsch et al (1985) Science 227:1229-1231). Generally, the Agrobacterium transformation system is used to engineer dicotyledonous plants (Bevan et al (1982) Ann. Rev. Genet 16:357-384; Rogers et al (1986) Methods Enzymol. 118:627-641). The Agrobacterium transformation system may also be used to transform, as well as transfer, DNA to monocotyledonous plants and plant cells. See Hernalsteen et al (1984) EMBO J 3:3039-3041; Hooykass-Van Slogteren et al (1984) Nature 311:763-764; Grimsley et al (1987) Nature 325:1677-179; Boulton et al (1989) Plant Mol. Biol. 12:31-40; and Gould et al (1991) Plant Physiol. 95:426-434.
Alternative gene transfer and transformation methods include, but are not limited to, protoplast transformation through calcium-, polyethylene glycol (PEG)- or electroporation-mediated uptake of naked DNA (see Paszkowski et al. (1984) EMBO J3:2717-2722, Potrykus et al. (1985) Molec. Gen. Genet. 199:169-177; Fromm et al. (1985) Proc. Nat. Acad. Sci. USA 82:5824-5828; and Shimamoto (1989) Nature 338:274-276) and electroporation of plant tissues (D'Halluin et al. (1992) Plant Cell 4:1495-1505). Additional methods for plant cell transformation include microinjection, silicon carbide mediated DNA uptake (Kaeppler et al. (1990) Plant Cell Reporter 9:415-418), and microprojectile bombardment (see Klein et al. (1988) Proc. Nat. Acad. Sci. USA 85:4305-4309; and Gordon-Kamm et al. (1990) Plant Cell 2:603-618).
The disclosed methods and compositions can be used to insert exogenous sequences into a predetermined location in a plant cell genome. This is useful inasmuch as expression of an introduced transgene into a plant genome depends critically on its integration site. Accordingly, genes encoding, e.g., nutrients, antibiotics or therapeutic molecules can be inserted, by targeted recombination, into regions of a plant genome favorable to their expression.
Transformed plant cells which are produced 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” in 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, organs, pollens, embryos or parts thereof. Such regeneration techniques are described generally in Klee et al (1987) Ann. Rev. of Plant Phys. 38:467-486.
Nucleic acids introduced into a plant cell can be used to confer desired traits on essentially any plant. A wide variety of plants and plant cell systems may be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present disclosure and the various transformation methods mentioned above. In preferred embodiments, target plants and plant cells for engineering include, but are not limited to, those monocotyledonous and dicotyledonous plants, such as crops including grain crops (e.g., wheat, maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce, spinach); flowering plants (e.g., petunia, rose, chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plants used in phytoremediation (e.g., heavy metal accumulating plants); oil crops (e.g., sunflower, rape seed) and plants used for experimental purposes (e.g., Arabidopsis).
Thus, the disclosed methods and compositions have use over a broad range of plants, including, but not limited to, species from the genera Asparagus, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucurbita, Daucus, Glycine, Hordeum, Lactuca, Lycopersicon, Malus, Manihot, Nicotiana, Oryza, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Solanum, Sorghum, Triticum, Vitis, Vigna, and Zea. One of skill in the art will recognize that after the expression cassette 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.
A transformed plant cell, callus, tissue or plant may be identified and isolated by selecting or screening the engineered plant material for traits encoded by the marker genes present on the transforming DNA. For instance, selection may be performed by growing the engineered plant material on media containing an inhibitory amount of the antibiotic or herbicide to which the transforming gene construct confers resistance. Further, transformed plants and plant cells may also be identified by screening for the activities of any visible marker genes (e.g., the .beta.-glucuronidase, luciferase, B or C1 genes) that may be present on the recombinant nucleic acid constructs. Such selection and screening methodologies are well known to those skilled in the art.
Physical and biochemical methods also may be used to identify plant or plant cell transformants containing inserted gene constructs. These methods include but are not limited to: 1) Southern analysis or PCR amplification for detecting and determining the structure of the recombinant DNA insert; 2) Northern blot, S1 RNase protection, primer-extension or reverse transcriptase-PCR amplification for detecting and examining RNA transcripts of the gene constructs; 3) enzymatic assays for detecting enzyme or ribozyme activity, where such gene products are encoded by the gene construct; 4) protein gel electrophoresis, Western blot techniques, immunoprecipitation, or enzyme-linked immunoassays, where the gene construct products are proteins. Additional techniques, such as in situ hybridization, enzyme staining, and immunostaining, also may be used to detect the presence or expression of the recombinant construct in specific plant organs and tissues. The methods for doing all these assays are well known to those skilled in the art.
Effects of gene manipulation using the methods disclosed herein can be observed by, for example, northern blots of the RNA (e.g., mRNA) isolated from the tissues of interest. Typically, if the amount of mRNA has increased, it can be assumed that the corresponding endogenous gene is being expressed at a greater rate than before. Other methods of measuring gene and/or CYP74B activity can be used. Different types of enzymatic assays can be used, depending on the substrate used and the method of detecting the increase or decrease of a reaction product or by-product. In addition, the levels of and/or CYP74B protein expressed can be measured immunochemically, i.e., ELISA, RIA, EIA and other antibody based assays well known to those of skill in the art, such as by electrophoretic detection assays (either with staining or western blotting). The transgene may be selectively expressed in some tissues of the plant or at some developmental stages, or the transgene may be expressed in substantially all plant tissues, substantially along its entire life cycle. However, any combinatorial expression mode is also applicable.
The present disclosure also encompasses seeds of the transgenic plants described above wherein the seed has the transgene or gene construct. The present disclosure further encompasses the progeny, clones, cell lines or cells of the transgenic plants described above wherein said progeny, clone, cell line or cell has the transgene or gene construct.
Delivery Vehicles
An important factor in the administration of polypeptide compounds, such as TAL-cleavage domain fusion protein, is ensuring that the polypeptide has the ability to traverse the plasma membrane of a cell, or the membrane of an intra-cellular compartment such as the nucleus. Cellular membranes are composed of lipid-protein bilayers that are freely permeable to small, nonionic lipophilic compounds and are inherently impermeable to polar compounds, macromolecules, and therapeutic or diagnostic agents. However, proteins and other compounds such as liposomes have been described, which have the ability to translocate polypeptides such as TAL-cleavage domain fusion proteins across a cell membrane.
For example, “membrane translocation polypeptides” have amphiphilic or hydrophobic amino acid subsequences that have the ability to act as membrane-translocating carriers. In one embodiment, homeodomain proteins have the ability to translocate across cell membranes. The shortest internalizable peptide of a homeodomain protein, Antennapedia, was found to be the third helix of the protein, from amino acid position 43 to 58 (see, e.g., Prochiantz, Current Opinion in Neurobiology 6:629-634 (1996)). Another subsequence, the h (hydrophobic) domain of signal peptides, was found to have similar cell membrane translocation characteristics (see, e.g., Lin et al., J. Biol. Chem. 270:14255-14258 (1995)).
Examples of peptide sequences which can be linked to a protein, for facilitating uptake of the protein into cells, include, but are not limited to: an 11 amino acid peptide of the tat protein of HIV; a 20 residue peptide sequence which corresponds to amino acids 84-103 of the p16 protein (see Fahraeus et al., Current Biology 6:84 (1996)); the third helix of the 60-amino acid long homeodomain of Antennapedia (Derossi et al., J. Biol. Chem. 269:10444 (1994)); the h region of a signal peptide such as the Kaposi fibroblast growth factor (K-FGF) h region (Lin et al., supra); or the VP22 translocation domain from HSV (Elliot & O'Hare, Cell 88:223-233 (1997)). Other suitable chemical moieties that provide enhanced cellular uptake may also be chemically linked to ZFPs. Membrane translocation domains (i.e., internalization domains) can also be selected from libraries of randomized peptide sequences. See, for example, Yeh et al. (2003) Molecular Therapy 7(5):S461, Abstract #1191.
Toxin molecules also have the ability to transport polypeptides across cell membranes. Often, such molecules (called “binary toxins”) are composed of at least two parts: a translocation/binding domain or polypeptide and a separate toxin domain or polypeptide. Typically, the translocation domain or polypeptide binds to a cellular receptor, and then the toxin is transported into the cell. Several bacterial toxins, including Clostridium perfringens iota toxin, diphtheria toxin (DT), Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus anthracis toxin, and pertussis adenylate cyclase (CYA), have been used to deliver peptides to the cell cytosol as internal or amino-terminal fusions (Arora et al., J. Biol. Chem., 268:3334-3341 (1993); Perelle et al., Infect. Immun., 61:5147-5156 (1993); Stennark et al., J. Cell Biol. 113:1025-1032 (1991); Donnelly et al., PNAS 90:3530-3534 (1993); Carbonetti et al., Abstr. Annu Meet. Am. Soc. Microbiol. 95:295 (1995); Sebo et al., Infect. Immun. 63:3851-3857 (1995); Klimpel et al., PNAS U.S.A. 89:10277-10281 (1992); and Novak et al., J. Biol. Chem. 267:17186-17193 1992)).
Such peptide sequences can be used to translocate TAL-cleavage domain fusion proteins across a cell membrane. TAL-cleavage domain fusion proteins can be conveniently fused to or derivatized with such sequences. Typically, the translocation sequence is provided as part of a fusion protein. Optionally, a linker can be used to link the TAL-cleavage domain fusion protein and the translocation sequence. Any suitable linker can be used, e.g., a peptide linker.
Targeted cleavage and targeted recombination can also be used to alter non-coding EBE sequences (e.g., regulatory sequences such as promoters, enhancers, initiators, terminators, splice sites) to alter the levels of expression of a gene product. Such methods can be used, for example, for therapeutic purposes, functional genomics and/or target validation studies.
The invention also provides methods for introducing the modified EBEs of the present invention into plants by crossing a plant which lacks the modified EBE with a plant that has the modified EBE, selfing the resulting generations and then selecting the plants exhibiting bacterial blight tolerance.
In another aspect, the invention provides a method for producing a hybrid seed comprising crossing a first plant parent with a second plant parent and harvesting the resultant hybrid seed, wherein either one or both parents contain modified EBEs. The hybrid seeds, plant and parts thereof produced by such method are also part of the invention.
In another aspect, the present invention provides for further single gene converted plants with modified EBEs. The desired further single transferred gene may preferably be a dominant or recessive allele. Preferably, the further single transferred gene will confer such traits as herbicide resistance, insect resistance, resistance for bacterial, fungal, or viral disease, male fertility, male sterility, enhanced nutritional quality, and industrial usage. The single gene may be a naturally occurring gene or a transgene introduced through genetic engineering techniques.
In another aspect, the present invention provides regenerable cells for use in tissue culture of a plant containing a modified EBE. The tissue culture will preferably be capable of regenerating plants having the physiological and morphological characteristics of the foregoing plant, and of regenerating plants having substantially the same genotype as the foregoing plant. Preferably, the regenerable cells in such tissue cultures will be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, pistils, roots, root tips, flowers, seeds, panicles or stems. Still further, the present invention provides plants regenerated from the tissue cultures of the invention.
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. Thus, many modifications and other embodiments of the invention will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.










TABLE OF SEQUENCES

















Wild type EBE sequence
nucleotide
SEQ ID NO: 1


−55 mutant
nucleotide
SEQ ID NO: 2


−32 mutant
nucleotide
SEQ ID NO: 3


−18 mutant
nucleotide
SEQ ID NO: 4


−9 mutant
nucleotide
SEQ ID NO: 5


−5 mutant
nuceotide
SEQ ID NO: 6


−4 mutant
nucleotide
SEQ ID NO: 7


−3 mutant
nucleotide
SEQ ID NO: 8


+9 mutant
nucleotide
SEQ ID NO: 9


−49 mutant
nucleotide
SEQ ID NO: 10


−8 mutant
nucleotide
SEQ ID NO: 11


−6 mutant
nucleotide
SEQ ID NO: 12


−5 mutant A
nucleotide
SEQ ID NO: 13


−4 mutant A
nucleotide
SEQ ID NO: 14


+2 mutant
nucleotide
SEQ ID NO: 15


AvrXa7 EBE
nucleotide
SEQ ID NO: 16


PthXo3 EBE
nucleotide
SEQ ID NO: 17


Os12N3 EBE
nucleotide
SEQ ID NO: 18


Os8N3 EBE
nucleotide
SEQ ID NO: 19


Overlapping AvrXa7
nucleotide
SEQ ID NO: 20


and PthXo3 EBE


dTALEN R1
nucleotide
SEQ ID NO: 21


dTALEN R2
nucleotide
SEQ ID NO: 22


dTALEN L1
nucleotide
SEQ ID NO: 23


dTALEN R1
amino acid
SEQ ID NO: 24


dTALEN R2
amino acid
SEQ ID NO: 25


dTALENL1
amino acid
SEQ ID NO: 26


forward primer of ~550 bp
nucleotide
SEQ ID NO: 27


with the target sites


reverse primer of ~550 bp
nucleotide
SEQ ID NO:28


with the target sites


internal sequencing
nucleotide
SEQ ID NO: 29


primer


Os11N3 primer
nucleotide
SEQ ID NO: 30


OS11N3 primer
nucleotide
SEQ ID NO: 31


Os04g19960 primer
nucleotide
SEQ ID NO: 32


Os04g19960 primer
nucleotide
SEQ ID NO: 33


TFIIAγ5 primer
nucleotide
SEQ ID NO: 34


TFIIAγ5 primer
nucleotide
SEQ ID NO: 35


SWT11-TALEN-L
nucleotide
SEQ ID NO: 36


SWT11-TALEN-L
amino acid
SEQ ID NO: 37


SWT11-TALEN-R
nucleotide
SEQ ID NO: 38


SWT11-TALEN-R
amino acid
SEQ ID NO: 39


SWT11-F primer
nucleotide
SEQ ID NO: 40


SWT11-R primer
nucleotide
SEQ ID NO: 41


PthXo1 EBE
nucleotide
SEQ ID NO: 42


Wild type OS8N3 promoter
nucleotide
SEQ ID NO: 43


Os8N3 −6
nucleotide
SEQ ID NO: 44


Os8N3 −8
nucleotide
SEQ ID NO: 45


Os8N3 −21
nucleotide
SEQ ID NO: 46


Os8N3 −14
nucleotide
SEQ ID NO: 47


Os11N3 −4b
nucleotide
SEQ ID NO: 48


SWT11-TALEN-L RVD
amino acid
SEQ ID NO: 49


SWT11-TALEN-L RVD target
nucleotide
SEQ ID NO: 50


SWT11-TALEN-R RVD
amino acid
SEQ ID NO: 51


SWT11-TALEN-R RVD target
nucleotide
SEQ ID NO: 52









The following examples are offered by way of illustration and not by way of limitation.
EXAMPLE 1
Transcription activator-like effectors (TALEs) of Xanthomonas oryzae pv. oryzae (Xoo) contribute to pathogen virulence through transcriptionally activating the rice disease susceptibility (S) genes. TALE nucleases (TALENs), the hybrid proteins of native or customized TAL effectors and the DNA cleavage domain of FokI, have been used to create intact yeast and animals whose genomes contain site-specific modifications. TALEN-mediated genome editing has not been demonstrated in plants. Here we exploit the paradigm of TALE/S-gene interaction using TALENs to edit the S gene in a way that renders it no longer responsive to the cognate TAL effector and, thus, confers resistance to bacterial blight of rice, a devastating disease in a crop that feeds half of the world's population.
We sought to target Os11N3, a developmentally regulated S gene, whose induction by TAL effector AvrXa7 or PthXo3 is required for bacterial blight disease in rice. The Os11N3 promoter contains a region wherein the effector binding element (EBE) for AvrXa7, EBE for PthXo3 and the TATA box of the promoter overlap (FIG. 1A). Two pairs of TALENs (Pair 1 and Pair 2) were deployed to induce mutations in the overlapping EBEs of the Os11N3 promoter and thus to interfere with the interaction between Os11N3 and AvrXa7 as well as PthXo3 in rice (FIG. 1A). The TALEs used for nuclease fusions included the AvrXa7 recognition region and three designer TALEs whose central 24 repeat units for recognition of 24 nucleotides in each TALEN were custom-made using our modular assembly method (FIG. 2).
One of the paired nuclease genes was constructed under the 35S promoter of cauliflower mosaic virus and the other under the maize ubiquitin 1 promoter in a single plasmid (FIG. 3). Rice embryonic cells were transformed for nuclease gene expression through Agrobacterium tumefaciens; individual transformant cells were selected, propagated and regenerated to transgenic plants (T0). The progeny (T1) of T0 plants were analyzed for site-specific sequence alterations by sequencing the PCR-amplified genomic DNA in the Os11N3 promoter. For Pair 1 nucleases, about two-thirds (63%) of the randomly selected T1 plants (n=30) carried a mono- or bi-allelic mutation from two independent transformation experiments (FIGS. 1B and 1C); about half (48%) of twenty eight T1 plants contained mutations that were induced by Pair 2 TALENs (FIG. 1D). In total, at least 14 distinct mutations including 6 in homozygous lines were detected in 53 T1 plants, and the majority of the mutations were small region deletions, all of which left the TATA box intact, with the exception of two deletions that co-existed with a wild type allele (FIG. 1E and FIG. 4). All mutant plants were morphologically normal compared to the segregating wild type plants.
We next investigated Os11N3 inducibility and susceptibility to the Xoo strain, which is dependent on AvrXa7 for virulence, in plants that were homozygous for mutations in the EBEs for AvrXa7 and PthXo1. As expected, the inducibility of Os11N3 by AvrXa7 was abolished in plants homozygous for the 9, 5, or 4 bp deletion or the 9 bp insertion; the plants also showed concomitant gain of resistance (health vs. curling and dry leaves) to the Xoo strain (FIGS. 1F and 1G). The loss of induction is specific to Os11N3 as the induction of Os04g19960, a transposon coding gene “collaterally” targeted by AvrXa7, was not affected (FIG. 1F). Bacterial infection assays on other T1 plants (n=627) generated from Experiment 1 (Pair 1) and not previously genotyped showed  ̃49% plants with resistance to the Xoo strain.
Taken together, the results demonstrate that the TALENs are highly capable of mediating site-specific genetic modifications in rice. Based on our results, this TALEN-based technology could be applicable to other plant species and holds great promise in facilitating genome-enabled research and crop improvement.
Materials and Methods
TALEN Design and Construction.
Native AvrXa7 with transcription activation domain truncated was used for fusion to the wild type FokI DNA cleavage domain. Designer TALENs were made using our previously described “modular assembly” method that involved four basic repeats for TAL effector DNA binding domain and the wild type FokI DNA cleavage domain. All four TALENs used the complete N-terminus of TAL effectors; another designer TALEN, like AvrXa7-FokI, used a C-terminus truncated TAL effector. DNA sequences for the open reading frames of TALENs are provided in FIG. 4.
Construction of TALEN Expression Plasmids and Rice Transformation.
Two promoters used to express the paired TALEN genes were the maize ubiquitin 1 promoter (ubi1) and the 35S promoter of cauliflower mosaic virus (35S). Both promoters were modified at the cloning sites (more detail is available upon request). One of the paired TALEN genes was cloned downstream of the 35S promoter by BamHI and SpeI sites in pCAMBIA1300 backbone (Cambia, Australia) and the other under ubi1 promoter also at BamHI and SacI sites. The ubi1-TALEN gene expression cassette was cut out with HindIII and moved into pCAMBIA 1300 derived plasmid that already contained the 35S-TALEN gene expression cassette. The resultant plasmids were mobilized into Agrobacterium tumefaciens strain EHA105 by electroporation. Agrobacterium-mediated transformation of the rice cultivar Kitake was conducted according to the protocol as described.
Sequencing Analysis of Genomic Regions in Os11N3 Targeted by TALENs.
Individual T1 plants were randomly selected for genomic DNA extraction using the CTAB method as previously described. Forward primer, 5′-TCCCTTAACTAGGACAACTTGGA-3′ (SEQ ID NO:27), and reverse primer, 5′-CCGGATCCAGCCATTGCAGCAAGATCTTG-3′ (SEQ ID NO:28), were used to amplify a region of  ̃550 bp with the preselected target sites located in the middle. The PCR products from individual plants were sequenced using an internal primer, 5′-CATGGCTGTGATTGATCAGG-3′ (SEQ ID NO:29). Each sequencing chromatogram was manually analyzed for polymorphisms within a trace.
Quantitative RT-PCR Analysis of Os11N3 Inducibility with AvrXa7.
Bacterial inoculums with optical density of 1.0 at 600 nm (OD600) were infiltrated by using needleless syringe into rice leaves as described. The bacterial strains are Xoo strain PXO99ME2 (hereinafter as ME2), a PXO99 derivative strain with loss of TAL effector pthXo1 and concomitant loss of strain virulence, and ME2(avrXa7), an ME2 transformant of avrXa7 gene with regain of strain virulence and Os11N3 gene induction. Total RNA of the inoculated portion of leaves was extracted using TRI reagent from Ambion 24 hours after inoculation, and RNA concentration and quality were measured using an ND-1000 Nanodrop spectrophotometer (Nanodrop Technologies). One microgram of RNA from each strain inoculation was treated with amplification grade DNase 1 (Invitrogen) followed by cDNA synthesis using the iScript cDNA synthesis kit (Bio-Rad). cDNA derived from 25 ng of total RNA was used for detection of gene induction by AvrXa7 using semi-quantitative PCR. In addition to inducing Os11N3, AvrXa7 also “collaterally” induced another rice gene Os04g19960, which encodes a putative retrotransposon protein, but is not associated with disease susceptibility in rice (our unpublished data). Gene-specific primers for Os11N3 are 5′-GAGAAGAAGGTAGCTGCATGAGTG-3′ (SEQ ID NO:30) and 5′-TCATGGAAGGAACCCTTACAGGTTG-3′ (SEQ ID NO:31), while primers for Os04g19960 are 5′-AGAAGGCGTAGGCATTCACAT-3′ (SEQ ID NO:32) and 5′-ACATTAACACAGCACACGTCAAC-3′ (SEQ ID NO:33). The rice general transcription factor TFIIAγ5 expression was used as an internal control with primers 5′-CTACTCAGCCAATAAATTGATAACTGC-3′ (SEQ ID NO:34) and 5′-CAATTTCTACTACTCATCGTTTAG-3′ (SEQ ID NO:35).
Disease Resistance Assay.
The fully expanded leaves of rice plants were inoculated by leaf tip clipping with scissors whose blades were immersed in bacterial suspensions (OD600=0.5) immediately prior to each clipping as described. Symptoms were scored by measuring lesion length 12-14 days after inoculation and categorized as resistance (R) if lesion lengths were shorter than 3 cm and susceptibility if longer than 5.
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EXAMPLE 2
In the previous example, two pairs of TALENs were employed to modify the overlapping EBEs in the promoter of Os11N3 (also called OsWEET14) bound by two TAL effectors AvrXa7 and PthXo3. Rice plants homozygous for the transmittable OsSWEET14 EBE mutations were resistant to AvrXa7- and PthXo3-dependent Xoo strains, and some rice lines were free of transgenes for TALENs and transformation marker. Those rice lines, however, were susceptible to the PthXo1-dependent Xoo strain PXO99 as the inducibility of OsSWEET11 by PthXo1 retained intact. Thus, we sought to address the questions such as whether TALENs could be useful to edit the function of other genes in rice, whether multiple (at least the two known) SWEET genes vulnerable to blight disease could be all edited sequentially to achieve broad disease resistance but not disrupt their normal functions.
Results
Design and Engineering of TALENs
DNA sequence at the PthXo1 EBE of OsSWEET11 was used for screening of two adjacent sites for a pair of TALENs to bind. A sequence of 17 bp starting at 86 bp upstream of the transcription initiation site (NCBI accession number AK070510) was chosen for assembling one TALEN (SWT11-TALEN-L), while a nearby sequence of 22 bp from the complementary strand was based to synthesize another TALEN (SWT11-TALEN-R). The two sites were separated by 19 bp, and the left site partially overlapped with the PthXo1 EBE (FIG. 5A). Four basic 34-amino acid repeats recognizing four nucleotides as represented by the RVD of NI for A, NG for T, NN for G, and HD for C were used to assemble the DNA binding domains of the two TALENs (FIG. 5B) (for DNA sequences see SEQ ID NOS: 36, 38; for amino acid sequences see SEQ ID NOS:37, 39). A modular assembly method modified from our previously developed was used to synthesize the repeats (Li, Huang et al. 2011). To avoid the detrimental induction of OsSWEET11 probably caused by TALEN consisting of a full-length TAL effector, the SWT11-TALEN-L contained a version of TAL effector with the C-terminal 40-amino acid transcription activation domain truncated, while the SWT11-TALEN-R contained the full-length TAL effector as DNA binding domain. The TALENs used a complete TAL effector N-terminus (SEQ ID NOS:36-39). The paired TALENs were active against the target site as measured in our yeast single strand annealing assay (SAA) (data not shown).
Construction of TALEN Gene Expression Cassettes
The two promoters used to express the paired TALEN genes were the maize ubiquitin 1 promoter (ubi1) and the 35S promoter of cauliflower mosaic virus (Christensen, Quail 1996, Odell, Nagy et al. 1985). The SWT11-TALEN-L gene was cloned downstream of the 35S promoter at BamHI and SpeI sites in the binary vector p35S-H3; the SWT11-TALEN-R gene was cloned under the control of the ubi1 promoter similarly but in vector pEH3. The ubi1-TALEN-R gene expression cassette was excised with HindIII and moved into the HindIII site of the binary plasmid containing the 35S-TALEN-L. The resultant plasmid was then transformed into Agrobacterium tumefaciens strain EHA105 by electroporation. Agrobacterium-mediated transformation of rice cultivar Kitake that already contained OsSWEET14 promoter mutation (FIG. 6A) was performed according to a previously described protocol (Hiei, Ohta et al. 1994).
TALENs Induced OsSWEET11 Promoter Mutations
The previously modified rice grew and produced normally, and the seeds could be easily induced into prolific embryonic cells for transformation. Individual transformant cells were selected, propagated and regenerated into whole plants (T0). Twelve independent transformant rice lines were obtained and were self-pollinated to produce seeds. The OsSWEET11 promoter regions were amplified using the polymerase chain reaction (PCR) with a set of primers (SWT11-F and SWT11-R) and the genomic DNA extracted from the T0 plant leaves. The PCR products were sequenced with the primer (SWT11-F). The primer sequences are provided in Table 1. Five out of twelve genotyped lines contained mono-allelic mutations (data not shown). Similarly, the T1 progeny of 4 mutant lines were genotyped (n=22). New mutations (deletions, or substitutions) including those in homozygous forms were revealed in some progeny (FIG. 6B). The mutations deleted partial or whole PthXo1 EBEs in the promoter of OsSWEET11. Representative mutations are shown in FIG. 6B. All mutant plants show no obvious growth defect compared to the wild type plants (data not shown).









TABLE 1






Primer
Sequence








SWT11-F
caccggtaccTGAGTGGTCAT



(SEQ ID NO: 40)
ACGTGTCATATTG


 



SWT11-R
CCGGATCCATTGCTACTGGTG



(SEQ ID NO: 41)
ATGAAGGT









TALEN-Induced OsSWEET11 Mutations Result in Xoo Resistance

The progeny (T1) homozygous for mutations of 6 bp, 8 bp, and 21 bp deletions within the OsSWEET11 promoter were inoculated with PXO99, a Xoo strain depended on its PthXo1 to induce OsSWEET11 for virulence (Yang, Sugio et al. 2006), and the symptom of leaf blight and lesion lengths were measured 14 days post inoculation in a standard disease assay. The mutants were strongly resistant to bacterial infection compared to the wild type plants in term of symptom (FIG. 7A) and lengths of lesion (FIG. 7B).
The contents of any patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entireties.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

## Claims

1. A genetically modified plant or plant cell which includes a TAL effector binding element (EBE) of a bacterial pathogen disease susceptibility gene that has been modified with a TALEN fusion protein to prevent induction of expression of said gene by a TAL effectors of essential virulence chosen from the group consisting of AvrXa7 and PthXo3; wherein said TALEN fusion includes one or more of SEQ ID NO: 24, 25 26 and 39, wherein said modified plant or plant cell retains normal plant growth and development; and wherein said disease susceptibility gene has been modified in the EBE region while the TATAAA box remains intact.

2. The plant cell of claim 1, wherein said TAL EBE is selected from the group consisting of SEQ ID NO: 16, 17, and 20.

3. The plant cell of claim 2, wherein said disease susceptibility gene is one of the N3 family of genes in rice.

4. The plant cell of claim 2, wherein said disease susceptibility gene is Os11N3.

5. The plant cell of claim 1, wherein said disease susceptibility gene is Os11N3.

6. The plant cell of claim 5, wherein said Os11N3 gene includes a modification in the PthXo3 and AVrXa7 overlapping EBE region, SEQ ID NO:20.

7. The plant cell of claim 6, wherein said modification is a deletion modification.

8. The plant cell of claim 7, wherein said modification includes a deletion of 9 nucleotides of SEQ ID NO:1 bases 27-35.

9. The plant cell of claim 7, wherein said modification includes deletion of 5 nucleotides or bases 27-31 from wild type SEQ ID NO:1.

10. The plant cell of claim 7, wherein said modification includes deletion of 4 nucleotides or bases 34-38 from wild type SEQ ID NO:1.

11. The plant cell of claim 6, wherein said modification is an insertion.

12. The plant cell of claim 11, wherein said modification is an insertion of 9 bases (gtttatata) between bases 37 and 38 from wild type SEQ ID NO:1.

13. The plant cell of claim 2, wherein said EBE region includes a nucleotide sequence selected from the group consisting of SEQ ID NO: 5, 6, 7 and 9.

14. The plant cell of claim 2, wherein said plant cell includes a heterologous nucleic acid expression construct including a nucleic acid sequence encoding a TALEN fusion protein including one or more of SEQ ID NO: 24, 25, 26 or 39.

15. The plant cell of claim 2 wherein said plant cell includes a heterologous fusion protein of SEQ ID NO: 24, 25, 26 or 39.

16. The plant of claim 2 wherein said plant is resistant to bacterial blight.

17. A genetically modified seed of the plant of claim 2.

18. A method of producing a plant that is resistant to bacterial blight caused by Xanthomonas oryzae pv. Oryzae comprising: introducing to said plant a modification in an EBE region of a disease susceptibility gene of Osl1N3 so that a TAL effector chosen from the group consisting of AvrXa7 and PthXo3 is prevented from inducing expression of the same and said modification allows for normal plant growth and development; wherein said modification leaves the TATAAA box intact; wherein said introducing is by transforming said plant with a heterologous nucleic acid selected from the group consisting of SEQ ID NO: 21, 22, 23, and 38 that encodes a TALEN fusion protein that creates modifications in a target EBE sequence.

19. The method of claim 18 further including the steps of crossing said modified EBE region plant with a second plant to produce progeny plants, and selecting plants exhibiting bacterial blight tolerance.

20. The method of claim 19 further including the steps of crossing said selected plants with said second plant to produce backcross progeny plants, selecting a first backcross progeny plant that has bacterial blight resistance to produce selected backcross progeny plants, repeating said crossing and selecting steps three or more times in succession to produce a backcross progeny plant that comprises bacterial blight resistance and essentially all of the physiological and morphological characteristics of said original second plant when grown in the same environmental conditions.

21. The method of claim 20, wherein
said TALEN fusion protein is SEQ ID NO: 24, 25, 26 or 39.

22. The method of claim 19, wherein said nucleic acid is
(a) SEQ ID NO: 21, 22, 23 or 38;
(b) a nucleic acid sequence which encodes SEQ ID NO: 24, 25, 26 or 39
(c) a nucleic acid sequence which has 90% or greater sequence identity to (a) or (b); wherein said TALEN fission protein creates modifications in a target EBE sequence yet retains the ability of the gene to provide normal plant growth.

23. The method of claim 22, wherein said nucleic acid includes a promoter sequence operably linked thereto.

24. A method for producing bacterial blight resistance in plants comprising: introducing a mutation in the EBE region of SEQ ID NO: 16, 17 or 20 of an Osl1N3 gene by transforming said plants with a heterologous nucleic acid selected from the group consisting of SEQ ID NO: 21, 22, 23, and 38, wherein said mutation leaves the TATAAA box intact, and further wherein said mutation prevents AvrXa7 and PthXo3 induction yet still allows for normal plant growth and development.

25. The method of claim 24 wherein said mutation is a deletion mutation.

26. The method of claim 25 wherein said mutation is an insertion mutation.

27. The method of claim 24 wherein said mutation occurs in the region of SEQ ID NO:20, the overlapping AvrXa7 and PthXo3 EBE.