WO2000032799A1 - Methods for transforming plastids - Google Patents

Abstract

An improved method is provided for the transformation of a plant cell plastid. The improved method allows for the increased efficiency of the foreign DNA penetrating the plastid membrane. The method generally involves the use of a plant tissue source having an altered plastid morphology in plastid transformation methods. The present invention finds use in plastid transformation methods for a wide variety of plant species.

Description

METHODS FOR TRANSFORMING PLASTIDS

FIELD OF THE INVENTION

This invention relates to the application of genetic engineering techniques to plants. More specifically, the invention relates to methods for the transformation of plant cell plastids.

BACKGROUND

The plastids of higher plants are an attractive target for genetic engineering.

Plant plastids (chloroplasts, amyloplasts, elaioplasts, chromoplasts, etc.) are the major biosynthetic centers that in addition to photosynthesis are responsible for production of industrially important compounds such as amino acids, complex carbohydrates, fatty acids, and pigments. Plastids are derived from a common precursor known as a proplastid and thus the plastids present in a given plant species all have the same genetic content. Plant cells contain 500-10,000 copies of a small 120-160 kilobase circular genome, each molecule of which has a large (approximately 25kb) inverted repeat. Thus, it is possible to engineer plant cells to contain up to 20,000 copies of a particular gene of interest which potentially can result in very high levels of foreign gene expression.

Current plastid transformation methods are inefficient, as such there is need for constructs and methods which improve plastid transformation.

SUMMARY OF THE INVENTION

By this invention, methods which allow for the improved transformation of a foreign DNA into plant cell plastids are provided. Such methods generally involve utilizing a plant tissue source which contains cells with an altered plastid morphology in the transformation methods. The alteration in the plant plastid morphology includes, inter alia, plastid size and number. By utilizing tissue derived from such plants in plastid transformation methods, efficiency of transformation of a foreign DNA into the plant cell plastid may be increased.

As exemplified herein, constructs useful for genetic engineering of plant cells to provide for a method of increasing plastid transformation efficiency are provided. The constructs include nucleic acid sequences coding for protein sequences involved in controlling division of plant cell organelles. The expression of such nucleic acid sequences in a plant cell provides for an altered number and/or size of the chloroplasts within the host cell.

DNA sequences, also referred to herein as polynucleotides, for use in transformation contain an expression construct comprising a promoter region which is functional in a plastid, and a DNA sequence encoding a gene involved in controlling the division of plant cell organelles.

Methods for the use of transformed plants with altered plastid morphology are described. Such methods include plant breeding or transformation methods to provide plant cells having both the nuclear and plastid constructs.

The present invention also provides methods for increasing the efficiency of chloroplast transformation. The method generally comprises transforming the plastids of a plant tissue which has been modified to have an altered number and/or size of plastids contained within the plant cell.

The present invention also provides a mechanism for enhancing the efficiency of chloroplast transformation in plant species. The present invention also provides methods for improving the selectability of plant comprising, transforming a plant cell source having an altered plastid morphology with a construct comprising a promoter functional in a plant cell plastid operably associated with a nucleic acid sequence encoding a selectable marker. Selectable markers of interest in the present invention include herbicide tolerance genes such as glyphosate tolerance genes, and antibiotic resistance genes. Glyphosate tolerance genes include the CP4 gene from Agrobacterium. Another aspect of the present invention provides methods for preparing a plant cell source with increased plastid transformation efficiency comprising, transforming a plant cell with a construct comprising a promoter functional in plant cell operably associated with a nucleic acid sequence encoding aFtsZ protein, Also considered part of this invention are the plants and plant cells obtained using the methods described herein.

DESCRIPTION OF THE FIGURES Figure 1 provides an amino acid sequence alignment of the Arabidopsis FtsZl

(SEQ ID NO:2), the Brassica FtsZl (SEQ ID NO:6), the tobacco FtsZl (SEQ ID NO:9), the Soybean FtsZl (SEQ ID NO:72) and the corn FtsZl (SEQ ID NO:73) protein sequences.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the subject invention, methods are provided which allow for the improved transformation of a foreign DNA into plant cell plastids. Such methods generally involve utilizing a plant cell source which contains an altered plant plastid morphology. By utilizing tissue derived from such plants in plastid transformation methods, efficiency of transformation of a foreign DNA into the plant cell plastid can be increased.

In one embodiment of the instant invention, plant tissue containing altered plant plastid morphology is used for plastid transformation methods. Such alterations in plant plastid morphology include, but are not limited to. alterations in the plastid size, shape and number in respect to a wild-type plastid morphology from the target plant cell. In general, a wild-type plastid morphology consists of small, round organelles contained within the plant cell, depending on the species. Furthermore, a plant cell typically contains between about 50 and about 100 plastids.

The plant tissue source used in plastid transformation methods of the present invention contains an increase in the size of the plastids contained in the plant cells. Such increases in the size of the plastids provides for a larger surface area for the foreign DNA to penetrate the plastid membrane during transformation.

The large plastids preferably contain approximately the same number of plastid genomes as would be contained in corresponding number of wild-type plastids. For example, in a wild-type plant cell containing 100 plastids per cell and 100 copies of the plastid genome in each plastid (a total of 10,000 copies of the plastid genome per cell), the corresponding mutant tissue source would preferably contain about the same number of plastid genomes, only contained in one, or several large plastid(s). Alternatively, a plant tissue source with an increased number of plastids, with a corresponding reduced size, can also find use in the plastid transformation methods of the present invention.

As is understood in the art, additional methods for obtaining plants with alterations in the plastid size and number are known. The skilled artisan will recognize that a number of methods are available for providing for an alteration in plastid cell division. Such methods are described, for example, by Strepp, et al. (1998) Proc. Natl. head. Sci. USA, 95:4368-4373.

Cell division, also referred to as cytokinesis, has been the focus of studies in many organisms such as bacterial, fungal, and animal cells. Division of bacterial cells occurs through the formation of an FtsZ ring (also referred to as a Z ring) at the site of division (Lutkenhaus, et al. (1997) Ann. Rev. Biochem. 66:93-116). The positioning and formation of the Z ring acts to further drive septation (cytokinesis). The ring is composed of a tubulin-like FtsZ protein which has GTPase activity. Mutations in the ftsZ gene in E. coli leads to the production of a temperature-sensitive filaments with regularly spaced nucleoids at certain temperatures (Lutkenhaus (1992) In Prokaryotic

Structure and Function: A New Perspective, ed. S Mohan, C Dow, pp 123-152. Cambridge: Cambridge Univ. Press). Such mutations in bacteria leads to the inability to divide correctly.

The plant cell plastid as well as the mitochondria are derived from prokaryotic ancestors, and thus, the division apparatus of these organelles resembles that of bacteria. Recently, identification offtsZ related sequences in Arabidopsis and Physcomitrella patens have been reported (Osteryoung, et al. (1995) Nature, 376:473-

SUBSTITUTΕ SHEET (RULE 26) 474; and Strepp, et al. (1998), supra). The protein encoded by the Arabidopsis ftsZ gene was found to be imported into the chloroplast and was therefore speculated to be a component of the plastid division machinery (Osteryoung, et al. (1995), supra). More recently, the involvement of FtsZ in plastid division was directly demonstrated. The disruption of the/tsZ gene in a lower plant, Physcomitrella patens, impeded plastid division, thereby giving rise to mutant cell lines with one or a few large plastids (Strepp, et al. (1998), supra).

The use of plants with an altered number and/or size of plastids containing one or few large plastids could therefore be used as targets for plastid transformation of any plant species. Such plants containing an altered size and/or number can be obtained using various methods, including mutagenesis, antisense suppression, or co- suppression. Methods for the mutagenesis of plant genomes are well known in the art, and include chemical, such as ethylmethane sulfonate (EMS) and nitrosoguanidine (NTG), as well as physical mutagenesis methods such as fast neutron bombardment. Other means for obtaining a plant source with an alteration in the size and/or number of plastids contained in the cell are also contemplated. For example, tissue for use in the transformation methods of the present invention can be obtained from plants grown in culture conditions which provide for such altered plastid content. For example, tissue obtained from plants grown in vitro under culture conditions in which inhibitors of bacterial cell division, such as 5,5'-bis-(8-anilino-l-naphtalenesulfonate) (Yu, et al. (1998) J. Biol Chem. 273: 10216-10222), are present, can be utilized as a cell source for the plastid transformation methods of the present invention.

In a preferred embodiment, such plants containing cells with an alteration in the size and/or number of plastids are generated by anti-sense expression of the FtsZ gene. Once plastid transformation is achieved and homoplasmic plants are identified, the anti-sense transgene can be eliminated by out-crossing and the wild-type condition of 50 to 100 plastids per cell restored. Similarly, plants regenerated from plastid transformed tissue containing an altered number and/or size of plastids from mutations can also be reverted to the wild-type plastid conditions using such out- crossing methods. In the case of the use of culture conditions for obtaining plant cells with an altered number and/or size of plastids, wild-type plastids can be obtained by releasing the tissue from such culture conditions.

In another embodiment of the present invention, novel nucleic acid sequences are provided which encode proteins related to proteins involved in bacterial cell and plastid division.

In particular, novel nucleic acid sequences from Arabidopsis, soybean, corn, Brassica are provided which encode FtsZ related proteins. Such nucleic acid sequences find use in the preparation of DNA constructs. Such constructs find use in the production of plants with an altered number and/or size of chloroplasts.

The skilled artisan will recognize that other DNA sequences useful for the production of plants with an altered number and/or size of chloroplasts are available in the art. The sequences include but are not limited to, ftsA,ftsL,ftsl.flsQ,ftsN, ftsW,ftsK (Lutkenhaus, et al. (1997) supra), and the arc genes (Pyke, et al. (1992) Plant Physiol. 99: 1005-1008; Pyke et al. (1994) Plant Physiol. 104:201-207; and Pyke (1997) Am. J. Botany 84: 1017-1027).

In order to obtain additional ftsZ sequences, a genomic or other appropriate library prepared from the candidate plant source of interest can be probed with conserved sequences from one or more plant and/or bacterial ftsZ sequence(s) to identify homologously related sequences. Positive clones can be analyzed by restriction enzyme digestion and/or sequencing. When a genomic library is used, one or more sequences can be identified providing both the coding region, as well as the transcriptional regulatory elements of theftsZ gene from such plant source. Probes can also be considerably shorter than the entire sequence. Oligonucleotides can be used, for example, but should be at least about 10, preferably at least about 15, more preferably at least 20 nucleotides in length. When shorter length regions are used for comparison, a higher degree of sequence identity is required than for longer sequences. Shorter probes are often particularly useful for polymerase chain reactions (PCR), especially when highly conserved sequences can be identified. (See, Gould, et al., PNAS USA (1989) 86:1934-1938.)

When longer nucleic acid fragments (>100 bp) are employed as probes, especially when using complete or large cDNA sequences, one can still screen with moderately high stringencies (for example using 50% formamide at 37°C with minimal washing) in order to obtain signal from the target sample with 20-50% deviation, i.e., homologous sequences. (For additional information regarding screening techniques see Beltz, et al., Meth. Enzymology (1983) 700:266-285). Another aspect of the present invention relates to isolated FtsZ polynucleotides. The polynucleotide sequences of the present invention include isolated polynucleotides that encode the polypeptides of the invention having a deduced amino acid sequence selected from the group of sequences set forth in the Sequence Listing and to other polynucleotide sequences closely related to such sequences and variants thereof.

The invention provides a polynucleotide sequence identical over its entire length to each coding sequence as set forth in the Sequence Listing. The invention also provides the coding sequence for the mature polypeptide or a fragment thereof, as well as the coding sequence for the mature polypeptide or a fragment thereof in a reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, pro-, orprepro- protein sequence. The polynucleotide can also include non-coding sequences, including for example, but not limited to, non- coding 5' and 3' sequences, such as the transcribed, untranslated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequence that encodes additional amino acids. For example, a marker sequence can be included to facilitate the purification of the fused polypeptide. Polynucleotides of the present invention also include polynucleotides comprising a structural gene and the naturally associated sequences that control gene expression. The invention also includes polynucleotides of the formula:

X-(R,)_n-(R₂)-(R₃)_n-Y wherein, at the 5' end, X is hydrogen, and at the 3' end, Y is hydrogen or a metal, Rj and R are any nucleic acid residue, n is an integer between 1 and 3000, preferably between 1 and 1000 and R is a nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from the group set forth in the Sequence Listing and preferably SEQ ID NOs: 1,3,5,7, 8, and 10-31. In the formula, R₂ is oriented so that its 5' end residue is at the left, bound to R_\, and its 3' end residue is at the right, bound to R₃. Any stretch of nucleic acid residues denoted by either R group, where R is greater than 1 , may be either a heteropolymer or a homopolymer, preferably a heteropolymer.

The invention also relates to variants of the polynucleotides described herein that encode for variants of the polypeptides of the invention. Variants that are fragments of the polynucleotides of the invention can be used to synthesize full-length polynucleotides of the invention. Preferred embodiments are polynucleotides encoding polypeptide variants wherein 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues of a polypeptide sequence of the invention are substituted, added or deleted, in any combination. Particularly preferred are substitutions, additions, and deletions that are silent such that they do not alter the properties or activities of the polynucleotide or polypeptide.

Further preferred embodiments of the invention that are at least 50%, 60%, or 70% identical over their entire length to a polynucleotide encoding a polypeptide of the invention, and polynucleotides that are complementary to such polynucleotides. More preferable are polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide encoding a polypeptide of the invention and polynucleotides that are complementary thereto. In this regard, polynucleotides at least 90% identical over their entire length are particularly preferred, those at least 95% identical are especially preferred. Further, those with at least 97% identity are highly preferred and those with at least 98% and 99% identity are particularly highly preferred, with those at least 99% being the most highly preferred.

Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptides encoded by the polynucleotides set forth in the Sequence Listing.

The invention further relates to polynucleotides that hybridize to the above- described sequences. In particular, the invention relates to polynucleotides that hybridize under stringent conditions to the above-described polynucleotides. As used herein, the terms "stringent conditions" and "stringent hybridization conditions" mean that hybridization will generally occur if there is at least 95% and preferably at least

97% identity between the sequences. An example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% formamide. 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/milliliter denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0. lx SSC at approximately 65°C. Other hybridization and wash conditions are well known and are exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, cold Spring Harbor, NY (1989), particularly Chapter 11.

The invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set for in the Sequence Listing under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence or a fragment thereof; and isolating said polynucleotide sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers as described herein. As discussed herein regarding polynucleotide assays of the invention, for example, polynucleotides of the invention can be used as a hybridization probe for RNA, cDNA, or genomic DNA to isolate full length cDNAs or genomic clones encoding a polypeptide and to isolate cDNA or genomic clones of other genes that have a high sequence similarity to a polynucleotide set forth in the Sequence Listing. Such probes will generally comprise at least 15 bases. Preferably such probes will have at least 30 bases and can have at least 50 bases. Particularly preferred probes will have between 30 bases and 50 bases, inclusive.

The coding region of each gene that comprises or is comprised by a polynucleotide sequence set forth in the Sequence Listing may be isolated by screening using a DNA sequence provided in the Sequence Listing to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to identify members of the library which hybridize to the probe. For example, synthetic oligonucleotides are prepared which correspond to the FtsZ EST sequences. The oligonucleotides are used as primers in polymerase chain reaction

(PCR) techniques to obtain 5' and 3' terminal sequence of FtsZ genes. Alternatively, where oligonucleotides of low degeneracy can be prepared from particular FtsZ peptides. such probes may be used directly to screen gene libraries for FtsZ gene sequences. In particular, screening of cDNA libraries inphage vectors is useful in such methods due to lower levels of background hybridization.

Typically, a FtsZ sequence obtainable from the use of nucleic acid probes will show 60-70% sequence identity between the target FtsZ sequence and the encoding sequence used as a probe. However, lengthy sequences with as little as 50-60% sequence identity may also be obtained. The nucleic acid probes may be a lengthy fragment of the nucleic acid sequence, or may also be a shorter, oligonucleotide probe. When longer nucleic acid fragments are employed as probes (greater than about 100 bp), one may screen at lower stringencies in order to obtain sequences from the target sample which have 20-50% deviation (i.e., 50-80% sequence homology) from the sequences used as probe. Oligonucleotide probes can be considerably shorter than the entire nucleic acid sequence encoding an FtsZ enzyme, but should be at least about 10, preferably at least about 15, and more preferably at least about 20 nucleotides. A higher degree of sequence identity is desired when shorter regions are used as opposed to longer regions. It may thus be desirable to identify regions of highly conserved amino acid sequence to design oligonucleotide probes for detecting and recovering other related FtsZ genes. Shorter probes are often particularly useful for polymerase chain reactions (PCR), especially when highly conserved sequences can be identified. (See, Gould, et al., PNAS USA (1989) 86: 1934-1938.).

Another aspect of the present invention relates to FtsZ polypeptides. Such polypeptides include isolated polypeptides set forth in the Sequence Listing, as well as polypeptides and fragments thereof, particularly those polypeptides which exhibit FtsZ activity and also those polypeptides which have at least 50%, 60% or 70% identity, preferably at least 80% identity, more preferably at least 90% identity, and most preferably at least 95% identity to a polypeptide sequence selected from the group of sequences set forth in the Sequence Listing, and also include portions of such polypeptides, wherein such portion of the polypeptide preferably includes at least 30 amino acids and more preferably includes at least 50 amino acids. "Identity", as is well understood in the art. is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. "Identity" can be readily calculated by known methods including, but not limited to, those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M. and Griffin, H.G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J Applied Math, 48:1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available programs. Computer programs which can be used to determine identity between two sequences include, but are not limited to, GCG (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); suite of five BLAST programs, three designed for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology, 12: 76-80 (1994); Birren, et al., Genome Analysis, 1: 543-559 (1997)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH, Befhesda, MD 20894; Altschul, S., et al, J. Mol. Bioi, 215:403-410 (1990)). The well known Smith Waterman algorithm can also be used to determine identity.

Parameters for polypeptide sequence comparison typically include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci USA 89: 10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4 A program which can be used with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison Wisconsin. The above parameters along with no penalty for end gap are the default parameters for peptide comparisons.

Parameters for polynucleotide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970) Comparison matrix: matches = +10; mismatches = 0

Gap Penalty: 50 Gap Length Penalty: 3

A program which can be used with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison Wisconsin. The above parameters are the default parameters for nucleic acid comparisons. The invention also includes polypeptides of the formula: X-(R,)_n-(R₂)-(R₃)_n-Y wherein, at the amino terminus, X is hydrogen, and at the carboxyl terminus, Y is hydrogen or a metal, Ri and R₃ are any amino acid residue, n is an integer between 1 and 1000, and R₂ is an amino acid sequence of the invention, particularly an amino acid sequence selected from the group set forth in the Sequence Listing and preferably SEQ ID NOs: 2,4,6, and 9. In the formula, R₂ is oriented so that its amino terminal residue is at the left, bound to Ri, and its carboxy terminal residue is at the right, bound to R₃. Any stretch of amino acid residues denoted by either R group, where R is greater than 1 , may be either a heteropolymer or a homopolymer, preferably a heteropolymer.

Polypeptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising a sequence selected from the group of a sequence contained in the Sequence Listing set forth herein . The polypeptides of the present invention can be mature protein or can be part of a fusion protein.

Fragments and variants of the polypeptides are also considered to be a part of the invention. A fragment is a variant polypeptide which has an amino acid sequence that is entirely the same as part but not all of the amino acid sequence of the previously described polypeptides. The fragments can be "free-standing" or comprised within a larger polypeptide of which the fragment forms a part or a region, most preferably as a single continuous region. Preferred fragments are biologically active fragments which are those fragments that mediate activities of the polypeptides of the invention, including those with similar activity or improved activity or with a decreased activity. Also included are those fragments that antigenic or immunogenic in an animal, particularly a human. Variants of the polypeptide also include polypeptides that vary from the sequences set forth in the Sequence Listing by conservative amino acid substitutions, substitution of a residue by another with like characteristics. In general, such substitutions are among Ala, Val, Leu and He; between Ser and Thr; between Asp and Glu; between Asn and Gin; between Lys and Arg; or between Phe andTyr. Particularly preferred are variants in which 5 to 10; 1 to 5; 1 to 3 or one amino acid(s) are substituted, deleted, or added, in any combination.

Variants that are fragments of the polypeptides of the invention can be used to produce the corresponding full length polypeptide by peptide synthesis. Therefore, these variants can be used as intermediates for producing the full-length polypeptides of the invention.

The polynucleotides and polypeptides of the invention can be used, for example, in the transformation of host cells, such as plant host cells, as further discussed herein.

The invention also provides polynucleotides that encode a polypeptide that is a mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids within the mature polypeptide (for example, when the mature form of the protein has more than one polypeptide chain). Such sequences can, for example, play a role in the processing of a protein from a precursor to a mature form, allow protein transport, shorten or lengthen protein half-life, or facilitate manipulation of the protein in assays or production. It is contemplated that cellular enzymes can be used to remove any additional amino acids from the mature protein.

A precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. The inactive precursors generally are activated when the prosequences are removed. Some or all of the prosequences may be removed prior to activation. Such precursor protein are generally called proprote ins. Homologous sequences are found when there is an identity of sequence and can be determined upon comparison of sequence information, nucleic acid or amino acid, or through hybridization reactions between a known FtsZ and a candidate source. Conservative changes, such as Glu/Asp, Val/Ile, Ser/Thr, Arg/Lys and Gln/Asn can also be considered in determining sequence homology. Typically, a lengthy nucleic acid sequence can show as little as 50-60% sequence identity, and more preferably at least about 70% sequence identity, between the target sequence and the given FtsZ sequence of interest excluding any deletions which can be present, and still be considered related. Amino acid sequences are considered homologous by as little as 25% sequence identity between the two complete mature proteins. (See generally, Doolittle, R.F., OF URFS and ORFS (University Science Books, CA, 1986.)

In addition, not only can sequences provided herein be used to identify homologous FtsZ sequences, but the resulting sequences obtained therefrom can also provide a further method to obtain FtsZ sequences from other plant and/or bacterial sources. In particular, PCR can be a useful technique to obtain related FtsZ sequences from sequence data provided herein. One skilled in the art will be able to design oligonucleotide probes based upon sequence comparisons or regions of typically highly conserved sequence.

Once the nucleic acid sequence is obtained, the transcription, or transcription and translation (expression), of the FtsZ sequence in a host cell is desired to produce a ready source of the enzyme and/or modify the number and/or size of the plastids found therein. Other useful applications can be found when the host cell is a plant host cell, in vitro and in vivo.

Nucleic acids (genomic DNA, plasmid DNA, cDNA, synthetic DNA, mRNA, etc.) encoding FtsZ or amino acid sequences of the purified enzymes, which permit design of nucleic acid probes facilitating the isolation of DNA coding sequences therefor, are known in the art and are available for use in the methods of the present invention. It is generally recognized to an artisan skilled in the field to which the present invention pertains that the nucleic acid sequences provided herein and the amino acid sequences derived therefrom can be used to isolate other potential FtsZ genes from GenBank using DNA and peptide search techniques generally known in the art. In addition to the sequences described in the present invention, DNA coding sequences useful in the present invention can be derived from algae, fungi, bacteria, plants, etc. Homology searches in existing databases using signature sequences corresponding to conserved nucleotide and amino acid sequences of FtsZ can be employed to isolate equivalent, related genes from other sources such as plants and microorganisms. Searches in EST databases can also be employed. Furthermore, the use of DNA sequences encoding enzymes functionally enzymatically equivalent to those disclosed herein, wherein such DNA sequences are degenerate equivalents of the nucleic acid sequences disclosed herein in accordance with the degeneracy of the genetic code, is also encompassed by the present invention. Demonstration of the functionality of coding sequences identified by any of these methods can be carried out by complementation of mutants of appropriate organisms, such as E. coli. The sequences of the DNA coding regions can be optimized by generesynthesis, based on codon usage, for maximum expression in particular hosts. The nucleic acid sequences which encode FtsZ can be used in various constructs, for example, as probes to obtain further sequences. Alternatively, these sequences can be used in conjunction with appropriate regulatory sequences to increase levels of the respective FtsZ sequence of interest in a host cell for recovery or study of the enzyme in vitro or in vivo or to decrease levels of the respective FtsZ sequence of interest for some applications when the host cell is a plant entity, including plant cells, plant parts (including but not limited to seeds, cuttings or tissues) and plants.

Thus, depending upon the intended use, the constructs can contain the nucleic acid sequence which encodes the entire FtsZ protein, or a portion thereof. For example, where antisense inhibition of a given FtsZ protein is desired, the entire FtsZ sequence is not required. Furthermore, where FtsZ constructs are intended for use as probes, it can be advantageous to prepare constructs containing only a particular portion of a FtsZ encoding sequence, for example a sequence which is discovered to encode a highly conserved FtsZ region. As discussed above, nucleic acid sequence encoding a plant or other FtsZ proteins of this invention can include genomic, cDNA or mRNA sequence. By "encoding" is meant that the sequence corresponds to a particular amino acid sequence either in a sense or anti-sense orientation. By "extrachromosomal" is meant that the sequence is outside of the plant genome of which it is naturally associated. By "recombinant" is meant that the sequence contains a genetically engineered modification through manipulation via mutagenesis, restriction enzymes, and the like. A cDNA sequence may or may not contain pre-processing sequences, such as transit peptide sequences or targeting sequences to facilitate delivery of the FtsZ protein to a given organelle or membrane location. The use of any such precursor FtsZ DNA sequence is preferred for uses in plant cell expression. A genomic FtsZ sequence can contain the transcription and translation initiation regions, introns, and/or transcript termination regions of the plant FtsZ, which sequences can be used in a variety of DNA constructs, with or without the FtsZ structural gene. Thus, nucleic acid sequences corresponding to the FtsZ sequences of this invention can also provide signal sequences useful to direct protein delivery into a particular organellar or membrane location, 5' upstream non-coding regulatory regions (promoters) having useful tissue and timing profiles, 3' downstream non-coding regulatory regions useful as transcriptional and translational regulatory regions, and may lend insight into other features of the gene.

Once the desired plant or other FtsZ nucleic acid sequence is obtained, it can be manipulated in a variety of ways. Where the sequence involves non-coding flanking regions, the flanking regions can be subjected to resection, mutagenesis, etc. Thus, transitions, transversions, deletions, and insertions can be performed on the naturally occurring sequence. In addition, all or part of the sequence can be synthesized. In the structural gene, one or more codons can be modified to provide for a modified amino acid sequence, or one or more codon mutations can be introduced to provide for a convenient restriction site or other purpose involved with construction or expression. The structural gene can be further modified by employing synthetic adapters, linkers to introduce one or more convenient restriction sites, or the like.

For the most part, the constructs will involve regulatory regions functional in plants which provide for altered size and number of plastids in a plant cell. The open reading frame, coding for the FtsZ protein. FtsZ-related protein or functional fragment thereof will be joined at its 5' end to a transcription initiation regulatory region such as the wild-type sequence naturally found 5' upstream to theFtsZ or FtsZ-related structural gene, or to a heterologous regulatory region from a gene naturally expressed in plant tissues. Examples of useful plant regulatory gene regions include those from T-DNA genes, such as nopaline oroctopine synthase, plant virus genes, such as CaMV 35S, or from native plant genes.

The DNA sequence encoding a plant or other FtsZ protein of this invention can be employed in conjunction with all or part of the gene sequences normally associated with the FtsZ. In its component parts, a DNA sequence encoding FtsZ is combined in a DNA construct having, in the 5' to 3' direction of transcription, a transcription initiation control region capable of promoting transcription and translation in a host cell, the DNA sequence encoding plant FtsZ and a transcription and translation termination region.

Potential host cells include both prokaryotic and eukaryotic cells. A host cell can be unicellular or found in a multicellar differentiated or undifferentiated organism depending upon the intended use. Cells of this invention can be distinguished by having a FtsZ sequence foreign to the wild-type cell present therein, for example, by having a recombinant nucleic acid construct encoding a FtsZ protein therein not native to the host species.

Depending upon the host, the regulatory regions will vary, including regions from viral, plasmid or chromosomal genes, or the like. For expression in prokaryotic or eukaryotic microorganisms, particularly unicellular hosts, a wide variety of constitutive or regulatable promoters can be employed. Expression in a microorganism can provide a ready source of the plant enzyme. Among transcriptional initiation regions which have been described are regions from bacterial and yeast hosts, such as E. coli, B. subtilis, Sacchromyces cerevisiae, including genes such as beta-galactosidase, T7 polymerase, tryptophan E and the like.

In a preferred embodiment, the constructs will involve regulatory regions functional in plants which provide for modified production of plant FtsZ, and. possibly, modification of the plant cell plastid. The open reading frame coding for the plant FtsZ or functional fragment thereof will be joined at its 5' end to a transcription initiation regulatory region. In embodiments wherein the expression of the FtsZ protein is desired in a plant host, the use of all or part of the complete plant FtsZ gene is desired; namely all or part of the 5' upstream non-coding regions (promoter) together with the structural gene sequence and 3' downstream non-coding regionscan be employed.

If a different promoter is desired, such as a promoter native to the plant host of interest or a modified promoter, i.e., having transcription initiation regions derived from one gene source and translation initiation regions derived from a different gene source, numerous transcription initiation regions are available which provide for a wide variety of constitutive or regulatable, e.g., inducible, transcription of the structural gene functions. The transcription/translation initiation regions corresponding to such structural genes are found immediately 5' upstream to the respective start codons. Among transcriptional initiation regions used for plants are such regions associated with the T-DNA structural genes such as for nopaline and mannopine synthases, the 19S and 35S promoters from CaMV, and the 5' upstream regions from other plant genes such as napin, ACP, SSU, PG, zein, phaseolin E, and the like. Enhanced promoters, such as double 35S, are also available for expression of FtsZ sequences. For such applications when 5' upstream non-coding regions are obtained from other genes regulated during seed maturation, those preferentially expressed in plant embryo tissue, such as ACP and napin-derived transcription initiation control regions, are desired. Such "seed-specific promoters" can be obtained and used in accordance with the teachings of issued U.S. Patent Numbers 5,608,152 and 5,530,194, which references are hereby incorporated by reference. Transcription initiation regions which are preferentially expressed in seed tissue, i.e., which are undetectable in other plant parts, are considered desirable for TAG modifications in order to minimize any disruptive or adverse effects of the gene product. Regulatory transcript termination regions can be provided in DNA constructs of this invention as well. Transcript termination regions can be provided by the DNA sequence encoding the plant FtsZ or a convenient transcription termination region derived from a different gene source, for example, the transcript termination region which is naturally associated with the transcript initiation region. Where the transcript termination region is from a different gene source, it will contain at least about 0.25 kb, preferably about 1-3 kb of sequence 3' to the structural gene from which the termination region is derived. Plant expression or transcription constructs having a plant FtsZ as the DNA sequence of interest for increased or decreased expression thereof can be employed with a wide variety of plant life, particularly, plant life involved in the production of vegetable oils for edible and industrial uses. Most especially preferred are temperate oilseed crops. Plants of interest include, but are not limited to, rapeseed (Canola and High Erucic Acid varieties), sunflower, safflower, cotton, soybean, peanut, coconut and oil palms, and corn. Depending on the method for introducing the recombinant constructs into the host cell, other DNA sequences can be required. Importantly, this invention is applicable to dicotyledenous and monocotyledenous species alike and will be readily applicable to new and/or improved transformation and regulation techniques.

The method of transformation is not critical to the instant invention; various methods of plant transformation are currently available. As newer methods are available to transform crops, they can be directly applied hereunder. For example, many plant species naturally susceptible to Agrobacterium infection can be successfully transformed via tripartite or binary vector methods oiAgrobacterium- mediated transformation. In addition, techniques of microinjection, DNA particle bombardment, and electroporation have been developed which allow for the transformation of various monocot and dicot plant species. In developing the DNA construct, the various components of the construct or fragments thereof will normally be inserted into a convenient cloning vector which is capable of replication in a bacterial host, e.g., E. coli. Numerous vectors exist that have been described in the literature. After each cloning, the plasmid can be isolated and subjected to further manipulation, such as restriction, insertion of new fragments, ligation, deletion, insertion, resection, etc., so as to tailor the components of the desired sequence. Once the construct has been completed, it can then be transferred to an appropriate vector for further manipulation in accordance with the manner of transformation of the host cell.

Normally, included with the DNA construct will be a structural gene having the necessary regulatory regions for expression in a host and providing for selection of transformant cells. The gene can provide for resistance to a cytotoxic agent, e.g. antibiotic, heavy metal, toxin, etc., complementation providing prototrophy to an auxotrophic host, viral immunity or the like. Depending upon the number of different host species in which the expression construct or components thereof are introduced, one or more markers can be employed, where different conditions for selection are used for the different hosts. A number of markers have been developed for use for selection of transformed plant cells, such as those which provide resistance to various antibiotics, herbicides, or the like. The particular marker employed is not essential to this invention, one or another marker being preferred depending on the particular host and the manner of construction.

As mentioned above, the manner in which the DNA construct is introduced into the plant host is not critical to this invention. Any method which provides for efficient transformation can be employed. Various methods for plant cell transformation include the use of Ti- or Ri-plasmids, microinjection, electroporation. DNA particle bombardment, liposome fusion, or the like. In many instances, it will be desirable to have the construct bordered on one or both sides by T-DNA, particularly having the left and right borders, more particularly the right border. This is particularly useful when the construct uses A. tumefaciens or A. rhizogenes as a mode for transformation, although the T-DNA borders can find use with other modes of transformation.

Once a transgenic plant is obtained which contains cells with altered numbers and/or sizes of chloroplasts, tissue containing such cells can then be used in plastid transformation experiments. For example, utilizing tissue containing cells with larger plastids provides for a larger target in plastid transformation methods, thus allowing for an increased probability of introduction of the foreign DNA into the plant cell plastid. The DNA sequences, or polynucleotides, for use in plastid transformation of this invention will contain a plastid expression construct generally comprising a promoter functional in a plant cell plastid. and a DNA sequence of interest to be expressed in the transformed plastid cells.

Constructs and methods for use in transforming the plastids of higher plants are described in Zoubenko et al. (Nuc Acid Res (1994) 22(19):3819-3824), Svab et al.

(Proc. Natl. Acad. Sc/.( 1990) 87:8526-8530 and Proc. Natl. Acad. 5c/.( 1993) 90:913- 917) and Staub et al. (EMBO J. (1993) 12:601-606). Constructs and methods for use in transforming plastids of higher plants to express DNA sequences under the control of a nuclearly encoded, plastid targeted T7 polymerase are described in U.S. Patent Number 5,576,198. The complete DNA sequences of the plastid genome of tobacco are reported by Shinozaki et al. (EMBO J. (1986) 5:2043-2049). Stable transformation of tobacco plastid genomes by particle bombardment is reported (Svab et.al. (1990), supra) and Svab et al. (1993), supra). The methods described therein can be employed to obtain plants homoplasmic for plastid expression constructs using the methods described herein. Briefly, such methods involve DNA bombardment of a target host explant, preferably from a tissue which is rich in metabolically active plastid organelles, such as green plant tissues including leaves, and cotyledons. The bombarded tissue is then cultured for ~2 days on a cell division promoting media. The plant tissue is then transferred to a selective media containing an inhibitory amount of the particular selective agent, as well as the particular hormones and other substances necessary to obtain regeneration for that particular plant species. For example, in the above publications and the examples provided herein, the selective marker is the bacterial aadA gene and the selective agent is spectinomycin. The aadA gene product allows for continued growth and greening of cells whose chloroplasts comprise the marker gene product. Cells which do not contain the marker gene product are bleached. The bombarded explants will form green shoots in approximately 3-8 weeks. Leaves from these shoots are then subcultured on the same selective media to ensure production and selection of homoplasmic shoots. As an alternative to a second round of shoot formation, the initial selected shoots can be grown to mature plants and segregation relied upon to provide transformed plants homoplastic for the inserted gene construct. The transformed plants so selected can then be analyzed to determine whether the entire plastid content of the plant has been transformed (homoplastic transformants). Typically, following two rounds of shoot formation and spectinomycin selection, approximately 50% of the transgenic plantlets analyzed are homoplastic as determined by Southern blot analysis of plastid DNA. These plantlets are selected for further cultivation, both for analysis of the transgenic plastid phenotype (where the nuclear viral polymerase expression construct is also present in the plastid transformant), or for use in methods to transform the viral polymerase construct into the nucleus of the transplastomic plants.

The methods of the present invention provide for a more efficient approach to obtaining homoplasmic plants. Wild-type plant cells typically contain 50 to 100 plastids per cell. However, once a transplastomic plant is obtained, the DNA sequence contained in the plant cell nucleus can be crossed away from the transplastomic cells. The DNA sequence transformed into the nucleus encoding for the alteration can be crossed away from the plant containing the transformed plastids. Once the DNA sequence has been crossed out, the plastids in the host plant cell can divide and revert back to normal (i.e. wild-type) plastid size and numbers. By applying the selective agent for which the plastid expression constructs provides resistance, cells containing a pure population of the plastids containing the foreign DNA can be obtained.

The vectors for use in plastid transformation preferably include means for providing a stable transfer of the plastid expression construct and selectable marker construct into the plastid genome. This is most conveniently provided by regions of homology to the target plastid genome. The regions of homology flank the construct to be transferred and provide for transfer to the plastid genome by homologous recombination, via a double crossover into the genome. The complete DNA sequence of the plastid genome of tobacco has been reported (Shinozaki et al.. EMBO J. (1986) 5:2043-2049). Complete DNA sequences of the plastid genomes from liverwort (Ohyama et al., Nature (1986) 322:572-574) and rice (Hiratsuka et al.. Mol. Gen. Genet. (1989) 277: 185-194), have also been reported.

Where the regions of homology are present in the inverted repeat regions of the plastid genome (known as IRA and IRB), two copies of the transgene are expected per transformed plastid. Where the regions of homology are present outside the inverted repeat regions of the plastid genome, one copy of the transgene is expected per transformed plastid. The regions of homology within the plastid genome are approximately lkb in size. Smaller regions of homology can also be used, and as little as 100 bp can provide for homologous recombination into the plastid genome.

However, the frequency of recombination and thus the frequency of obtaining plants having transformed plastids decreases with decreasing size of the homology regions. Examples of constructs comprising such regions of homology for tobacco plastid transformation are described in Svab et.al. (1990 supra) and Svab and Maliga (1993 supra). Regions useful for recombination into tobacco and Brassica plastid genomes are also described in the following examples. Similar homologous recombination and selection constructs can be prepared using plastid DNA from the target plant species. Other means of transfer to the plastid genome are also considered herein, such as by methods involving the use of transposable elements. For example, the constructs to be transferred into the plastid genome can be flanked by the inverted repeat regions from a transposable marker which functions in plant plastids. A DNA construct which provides for transient expression of the transposase required to transfer the target DNA into the plastids is also introduced into the chloroplasts. In this manner, a variety of phenotypes can be obtained in plants transformed with the same expression construct depending on positional effects which can result from insertion of the expression constructs into various locations on the plastid genome. Appropriate transposons for use in such plastic transformation methods include bacterial TnlO, bacteriophage Mu and various other known bacterial transposons.

The DNA sequence of interest in the plastid promoter expression constructs can be an encoding sequence which is oriented for expression of a particular structural gene, such that the protein encoded by the structural gene sequence is produced in the transformed plastid. In addition, the DNA sequence of interest can include a number of individual structural gene encoding regions such that an operon for expression of a number of genes from a single plastid promoter region is produced. Thus, it is possible to introduce and express multiple genes from an engineered or synthetic operon or from a pre-existing prokaryotic gene cluster. Such a method would allow large scale and inexpensive production of valuable proteins and fine chemicals in a particular desired plant tissue or a particular stage of development, depending upon the promoter used to drive nuclear expression of the specific viral polymerase. Such an approach is not practical by standard nuclear transformation methods since each gene must be engineered into a monocistron including an encoded transit peptide for plastid uptake and appropriate promoter and terminator signals. As a result, gene expression levels would be expected to vary widely between cistrons, and generation of a number of transgenic plant lines would be required. Ultimately crosses would be required to introduce all of these cistrons into one plant to get expression to the target biochemical pathway.

Alternatively, the DNA sequence of interest in the plastid construct can be a fragment of an endogenous plastid gene oriented such that an RNA complementary to the endogenous gene mRNA is produced in the transformed plastid. Such antisense constructs can be used to decrease the expression of the target plastid gene.

In order to provide a means of selecting the desired plant cells following plastid transformation, the polynucleotides for plastid transformation will also contain a construct which provides for expression of a marker gene. Expression of the marker gene product allows for selection of plant cells comprising plastid organelles which are expressing the marker protein. In the examples provided herein, a bacterial aadA gene is expressed under the regulatory control of chloroplast 5' promoter and 3' transcription termination regions. The use of such an expression construct for plastid transformation of plant cells has been described by Svab and Maliga (1993, supra). Expression of the aadA gene confers resistance to spectinomycin and streptomycin, and thus allows for the identification of plant cells expressing this marker gene. Selection for the aadA marker gene is based on identification of plant cells which are not bleached by the presence of streptomycin, or more preferably spectinomycin, in the plant growth medium. Other genes which encode a product involved in chloroplast metabolism can also be used as selectable markers. For example, genes which provide resistance to plant herbicides such as glyphosate, bromoxynil or imidazolinone can find particular use. Such genes have been reported by Stalker et al. (J. Biol. Chem. (1985) 260:4724-4728; glyphosate resistant EPSP), Stalker et al. (J. Biol. Chem. (1985) 263:6310-6314; bromoxynil resistant nitrilase gene), and Sathasivan et al. (Nucl. Acids Res. (1990) i °:2188; AHAS imidazolinone resistance gene).

The present invention also provides methods for obtaining a plastid transformed plant on medium containing glyphosate. At the initial event of transformation only a few plastids out of the many present in a plant cell are transformed and therefore are able to express glyphosate resistant marker gene product. The rest of the untransformed plastids within the cell remains vulnerable to the effect of glyphosate. Therefore, although the cell contains transformed plastids, it is unable to divide and sort out the transformed plastid resulting in lack of recovery of transformed callus tissue which would give rise to the transformed regenerants. Thus, any method that reduces plastid number to one or few within the cell has the potential to survive the effect of glyphosate and be useful as selectable marker for plastid transformation.

The following examples are provided by way of illustration and not by way of limitation.

EXAMPLES

Example 1: Identification of Plant ftsZ Sequences

In order to obtain a plant tissue source with an altered number and/or size of plastids using antisense and/or sense expression of the bacterial FtsZ plant homologues, public as well as proprietary sequence databases are queried for homologous sequences in soybean, rice, Arabidsopsis, corn and Brassica. Two types of plant FtsZ proteins have been previously identified in GenBank, type I FtsZ proteins exemplified by accession gil 1079731 (SEQ ID NO:32), appear to be imported into the plastid, while type II FtsZ proteins, exemplified by accession gil3608494

(SEQ ID NO:33) and gil683524 (SEQ ID NO:34), appear to remain in the cytoplasm. Homologs of both the type I FtsZ sequence as well as homologues of type II FtsZ genes are described below. The sequences used to search against the databases are: type I FtsZ homologue search was (SEQ ID NO:32), and for type IIFtsZ searches, (SEQ ID NO:33) is used.

Searches performed in proprietary databases containing sequences obtained from Arabidopsis identified DNA sequences which are related to the FtsZl sequence. The sequence of SEQ ID NO: l is identified as AtFtsZl. The deduced amino acid sequence encoded by SEQ ID NO: 1 is provided in SEQ ID NO:2. In addition, one sequence (SEQ ID NO:3) was identified as related to the FtsZ2 sequence. The deduced amino acid sequence encoded by SEQ ID NO:3 is provided in SEQ ID NO:4. Sequences were also identified in databases containing sequences obtained from Brassica. One sequence was identified as related to the Arabidopsis FtsZl sequence. Based on sequence alignments between the two sequences, approximately 170 amino acids were predicted to be missing from the Brassica sequence at the N- terminus. To obtain a full length coding sequence for the Brassica FtsZl (BnFtsZl) gene, RACE PCR using DNA obtained from Brassica leaves was performed using the primers SC258 (SEQ ID NO:35) and SC259 (SEQ ID NO:36). One reaction product was found to contain the most 5' sequence (SEQ ID NO:70) and was used to produce a full length sequence referred to as BnFtsZl (SEQ ID NO:5). The deduced amino acid sequence encoded by BnFtsZl is provided in SEQ ID NO:6)

A FtsZl homolog was also identified in tobacco with PCR using primers designed to the conserved amino acid domains of the Arabidopsis FtsZl sequence. The PCR primers used are identified as SC252 (SEQ ID NO:37), SC253 (SEQ ID NO:38), SC254 (SEQ ID NO:39) and SC255 (SEQ ID NO:40). The reaction products were cloned into TOPO TA (Invitrogen), and a single clone, referred to as xanthil-26- contig (SEQ ID NO:7), contained the most sequence. Additional primers were designed for use in RACE PCR to obtain full length coding sequence for the tobacco FtsZl homolog. For amplification of the 5' region, primers SC291 (SEQ ID NO:41) and SC292 (SEQ ID NO:42) were used, and for amplification of the 3' sequence, primers SC293 (SEQ ID NO:43) and SC294 (SEQ ID NO:44) were used. The PCR products were cloned in TOPO TA and sequenced. Clone xanftsZl-5'-15 (SEQ ID NO:71) was chosen to be the best for the 5' tobacco FtsZl sequence since it contained the greatest amount of 5' sequence and overlap with xanthil-26-contig. This sequence was combined with the xanthil-26-contig to produce xanFtsZl (SEQ ID NO:8). The deduced amino acid sequence is provided in SEQ ID NO:9.

FtsZ homolog sequences were identified in databases containing DNA sequences obtained from corn by BLAST searches using the Arabidopsis FtsZl and FtsZ2 amino acid sequences. Ten sequence were identified as related to these FtsZ sequences, provided in SEQ ID NOs: 10-19. The clones, when aligned, revealed six contigs, and the best representative clone for each were chosen for further analysis.

Sequence analysis of SEQ ID NO: 10 revealed a high homology to AtFtsZl, and was estimated to be missing 158 amino acids at the N-terminal end when compared to Arabidopsis FtsZl. Clone SEQ ID NO: 13 was found to overlap perfectly with SEQ ID NO: 10 for 153 nt at the 5 'end and in addition had 167 nt additional nt at the 5' end that had amino acid homology with the Arabidopsis FtsZl. However, this clone was also not predicted to encode the full-length FtsZ, and was still missing 113 amino acids at the N-terminal end when compared to Arabidopsis FtsZl. Interestingly, for clone SEQ ID NO: 13, its homology with SEQ ID NO:10, ends at position 167nt and diverges. This could either be indicative of the presence of intronic sequence or a new class of FtsZ protein. Primer SC321 (SEQ ID NO:45) was designed to pull out the missing maize FtsZl sequence by RACE PCR. Sequence analysis of SEQ ID NO: 18 revealed its high homology to FtsZ2, and was also predicted to not to be full-length and missing about 286 amino acids at the N-terminal end when compared to Arabidopsis FtsZ2. Primer SC322 (SEQ ID NO:46) was designed to pull out the missing maize FtsZ2 sequence by RACE PCR. Although SEQ ID NO; 14 and SEQ ID NO: 15 were identified with the highest BLAST scores with FtsZ2.

Soybean FtsZ homolog sequences were identified in databases by BLAST searches with Arabidopsis FtsZl and FtsZ2 amino acid sequences. Twelve sequences were obtained, and are provided in SEQ ID NOs:20-31. Sequence analysis of SEQ ID NO:20, SEQ ID NO:24 and SEQ ID NO:25 revealed high homology to FtsZl and none to be full-length when compared to Arabidopsis FtsZl. SEQ ID NO:25 had the longest sequence at the N-terminal end and is predicted to be missing 64 amino acids at the N-terminal when compared to Arabidopsis FtsZl sequence. Sequences of SEQ ID NO:20, SEQ ID NO:24 and SEQ ID NO:25 were used to correct the overlapping region. RACE PCR primers can now be designed to amplify the ends for obtaining a full length DNA sequence.

A sequence alignment between the Arabidopsis, Brassica, tobacco, soybean, and corn FtsZl protein sequences is provided in figure 1.

Example 2: Preparation of Plant Expression Constructs

2A. Nuclear Expression Constructs Constructs are prepared for transformation into a plant cell nucleus for alteration of the plastid size and/or number in the transformed plant cell. Constructs can be prepared to alter the plastids constitutively, or in a tissue specific manner, for example, in leaf tissue, or seed tissue. A plasmid containing the napin cassette derived from pCGN3223 (described in USPN 5,639,790, the entirety of which is incorporated herein by reference) was modified to make it more useful for cloning large DNA fragments containing multiple restriction sites, and to allow the cloning of multiple napin fusion genes into plant binary transformation vectors. An adapter comprised of the self annealed oligonucleotide of sequence

CGCGATTTAAATGGCGCGCCCTGCAGGCGGCCGCCTGCAGGGCGCGCCAT TTAAAT (SEQ ID NO:47) was ligated into the cloning vector pBC SK+ (Stratagene) after digestion with the restriction endonuclease BssHII to construct vector pCGN7765. Plamids pCGN3223 and pCGN7765 were digested with Notl and ligated together. The resultant vector, pCGN7770, contains the pCGN7765 backbone with the napin seed specific expression cassette from pCGN3223.

The cloning cassette, pCGN7787, essentially the same regulatory elements as pCGN7770, with the exception of the napin regulatory regions of pCGN7770 have been replaced with the double CAMV 35S promoter and the tml polyadenylation and transcriptional termination region.

A binary vector for plant transformation, pCGN5139, was constructed from pCGN1558 (McBride and Summerfelt, (1990) Plant Molecular Biology, 14:269-276). The polylinker of pCGN1558 was replaced as a HindIII/Asp718 fragment with a polylinker containing unique restriction endonuclease sites, Ascl, Pad, Xbal, Swal. BamHI,and Notl. The Asp718 and Hindlll restriction endonuclease sites are retained in pCGN5139.

A series of turbo binary vectors are constructed to allow for the rapid cloning of DNA sequences into binary vectors containing transcriptional initiation regions (promoters) and transcriptional termination regions. The plasmid pCGN8618 was constructed by ligating oligonucleotides 5'-

TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID NO:48) and 5'- TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC-3' (SEQ ID NO:49) into Sall/XhoI-digested pCGN7770. A fragment containing the napin promoter, polylinker and napin 3' region was excised from pCGN8618 by digestion with Asp718I; the fragment was blunt-ended by filling in the 5' overhangs with Klenow fragment then ligated into pCGN5139 that had been digested with Asp718I and Hindlll and blunt- ended by filling in the 5' overhangs with Klenow fragment. A plasmid containing the insert oriented so that the napin promoter was closest to the blunted Asp718I site of pCGN5139 and the napin 3' was closest to the blunted Hindlll site was subjected to sequence analysis to confirm both the insert orientation and the integrity of cloning junctions. The resulting plasmid was designated pCGN8622. The plasmid pCGN8619 was constructed by ligating oligonucleotides 5'-

TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC -3' (SEQ ID NO:50) and 5'- TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID NO:51) into Sall/XhoI-digested pCGN7770. A fragment containing the napin promoter, polylinker and napin 3' region was removed from pCGN8619 by digestion with Asp718I; the fragment was blunt-ended by filling in the 5' overhangs with Klenow fragment then ligated into pCGN5139 that had been digested with Asp718I and Hindlll and blunt- ended by filling in the 5' overhangs with Klenow fragment. A plasmid containing the insert oriented so that the napin promoter was closest to the blunted Asp7181 site of pCGN5139 and the napin 3' was closest to the blunted Hindlll site was subjected to sequence analysis to confirm both the insert orientation and the integrity of cloning junctions. The resulting plasmid was designated pCGN8623.

The plasmid pCGN8620 was constructed by ligating oligonucleotides 5'- TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGGAGCT -3' (SEQ ID NO:52) and 5'-CCTGCAGGAAGCTTGCGGCCGCGGATCC-3' (SEQ ID NO:53) into Sall/SacI-digested pCGN7787. A fragment containing the d35S promoter, polylinker and tml 3' region was removed from pCGN8620 by complete digestion with Asp718I and partial digestion with Notl. The fragment was blunt-ended by filling in the 5' overhangs with Klenow fragment then ligated into pCGN5139 that had been digested with Asp718I and Hindlll and blunt-ended by filling in the 5' overhangs with Klenow fragment. A plasmid containing the insert oriented so that the d35S promoter was closest to the blunted Asp718I site of pCGN5139 and the tml 3' was closest to the blunted Hindlll site was subjected to sequence analysis to confirm both the insert orientation and the integrity of cloning junctions The resulting plasmid was designated pCGN8624

The plasmid pCGN8621 was constructed by ligating oligonucleotides 5'- TCGACCTGCAGGAAGCTTGCGGCCGCGGATCCAGCT -3' (SEQ ID NO.54) and 5'-GGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID NO 55) into

Sall/SacI-digested pCGN7787. A fragment containing the d35S promoter, polylinker and tml 3' region was removed from pCGN8621 by complete digestion with Asp718I and partial digestion with Notl The fragment was blunt-ended by filling in the 5' overhangs with Klenow fragment then ligated into pCGN5139 that had been digested with Asp718I and Hindlll and blunt-ended by filling in the 5' overhangs with Klenow fragment A plasmid containing the insert oriented so that the d35S promoter was closest to the blunted Asp718I site of pCGN5139 and the tml 3' was closest to the blunted Hindlll site was subjected to sequence analysis to confirm both the insert orientation and the integrity of cloning junctions The resulting plasmid was designated pCGN8625.

The plasmid construct pCGN8640 is a modification of pCGN8624 described above. A 938bp Pstl fragment isolated from transposon Tn7 which encodes bacterial spectinomycin and streptomycin resistance (Fling et al. ( 1985), Nucleic Acids Research 13(19)-7095-7106), a determinant for E. coli and Agrobacteπum selection, was blunt ended with Pfu polymerase The blunt ended fragment was ligated into pCGN8624 that had been digested with Spel and blunt ended with Pfu polymerase The region containing the Pstl fragment was sequenced to confirm both the insert orientation and the integrity of cloning junctions

The spectinomycin resistance marker was introduced into pCGN8622 and pCGN8623 as follows A 7.7 Kbp Avrll-SnaBI fragment from pCGN8640 was ligated to a 10.9 Kbp Avrll-SnaBI fragment from pCGN8623 or pCGN8622, described above. The resulting plasmids were pCGN8641 and pCGN8643, respectively

The Arabidopsis FtsZl nucleotide sequence was used to construct the sense expression vector pCGN6495 for use in transformation of Arabidopsis. Brassica and tobacco. For this construct, the Arabidopsis ftsZl sequence was PCR amplified. To monitor protein expression of FtsZl in transformed lines, a c-myc tag (EQKLISEEDL (SEQ ID NO:56)), was translationally fused to FtsZl at the C-terminal end. The PCR amplification was done by first round of amplification with primers SC247 (SEQ ID NO:57) and SC260 (SEQ ID NO:58) followed by amplification with SC247 (SEQ ID NO:59) and SC261 (SEQ ID NO:60) using the product of the first amplification as the template DNA, using standard amplification parameters. The final amplification product, FtsZl/c-myc fusion was cloned in the nuclear transformation vector pCGN8624 to create pCGN6495, which was used to nuclear transform Arabidopsis. canola and tobacco using standard protocols.

The turbo vector pCGN8624 was used for the antisense constructs such that the antisense sequence is driven from d35S promoter. For Arabidopsis the coding sequence (from ATG to TAG) was amplified with primers SC248 (SEQ ID NO:61) and SC250 (SEQ ID NO:62) using AtFtsZl as template. For Brassica₁ primers SC276 (SEQ ID NO:63) and SC268 (SEQ ID NO:64) were used with PCR fragment SC3-1-1 (SEQ ID NO:70) as template DNA to generate aHindϊLVPstl fragment and cloned in pBSKS (Stratagene) to generate pCGN6528. Primer SC276 was designed to be located 140 bases downstream from ATG due to the presence of nonhomologous stretch of sequence compared to Arabidopsis FtsZl contained in the first 140 bases sequence fragment. The 3' half of the coding sequence was PCR amplified using primers SC269 (SEQ ID NO:65) and SC270 (SEQ ID NO:66) to produce aPstl/Notl fragment, and subsequently cloned in pCGN6528 to generate pCGN6529. The

HindlWPstl fragment containing BnFtsZl sequence (from 140b downstream of ATG to TAG) was cloned in turbo vector pCGN8624 to generate final transformation vectors pCGN6530 and pCGN6611. The HindllUNotl fragment containing BnFtsZl sequence was also cloned into pCGN8643 vector for seed-specific antisense FtsZl expression. For tobacco, primers SC305 and SC306 were designed to PCR amplify FtsZl sequence to produce a SseVNotl fragment using 5' RACE PCR library DNA made from leaf RNA, and cloned into TOPO TA2.1 to produce pCGN6565. The SseVNotl fragment from pCGN6565 was cloned in the turbo vector pCGN8624 to generate final transformation vector pCGN6566.

2A. Plastid Expression Constructs Constructs and methods for use in transforming the plastids of higher plants are described in Zoubenko et al. (Nuc Acid Res (1994) 22(19):3819-3824), Svab et al. (Proc. Natl. Acad. Sci.(l990) 87:8526-8530 and Proc. Natl. Acad. Sci.(\993) 90:913- 917) and Staub et al. (EMBO J. (1993) 12:601-606). Constucts and methods for use in transforming plastids of higher plants to express DNA sequences under the control of a nuclearly encoded, plastid targeted T7 polymerase are described in U.S. Patent Number 5,576,198. The complete DNA sequences of the plastid genome of tobacco are reported by Shinozaki et al. (EMBO J. (1986) 5:2043-2049).

A plastid expression construct, pMON49218, was constructed to express the synthetic CP4 sequence with the 14 amino acid GFP fusion from the promoter region of the lόSrDNA operon having the nuclear-encoded RNA polymerase region (PrrnPEP+NEP), and the terminator region from the plastid rpslό gene. The DNA sequence of the Prrn/NEP/GIOL:: 14aaGFP fusion SEQ ID NO:67.

Example 3: Plant Transformation And Analysis

Constructs for the expression of sense or antisense sequences are transformed into tobacco cells using the methods described by Ursin et. al. (1991) Plant Cell 3:583-591.

Transgenic tobacco plants containing the nuclear FtsZ constructs were analyzed for alterations in plastid morphology, including size and number of plastids present in the plant cell.

Fifty-eight initial transformants (Tl generation) obtained from transformation with FtsZl expression construct pCGN6495 were screened for the large plastid phenotype and divided into three categories. Thirty-four (34) lines contained less than 5 large plastids, 8 lines contained between 5-20 plastids and 16 lines more than 20 (wild-type# and more than wild-type#) plastids. One line, Nt6495-61, contained a single large plastid. The screening method involved examining isolated mesophyll protoplasts at

100X magnification under light microscope. The large plastid containing transgenic plants appear to be phenotypically indistinguishable from wild-type under culture and greenhouse conditions.

Estimation of plastid DNA copy number from several large plastid lines revealed no difference when compared to wild-type. Southern analysis was used to estimate transgene copy number in the large plastid lines and several lines with single integration events were identified. Western analysis of the large plastid lines with c- myc antibody confirmed expression of the introduced transgene (tagged by c-myc). T2 seeds were collected from selected plants from each of the three categories.

Example 4: Plastid Transformation and Analysis

Leaf material from three transgenic lines, Nt6495-30 (with <5plastids/cell), Nt6495-16 (with 5-20 plastids/cell) and Nt6495-69 (with 5-20 plastids/cell), were obtained for evaluation of plastid transformation efficiency and direct glyphosate selection. Plastid transformation vector pMON49218 which contains aadA gene for spectinomycin selection and GFP as a marker was used to bombard 15 leaf explants of each of the three transgenic lines. For each series of bombardment of the transgenic line 15 wild type control leaves were used. The order of bombardment for the transgenic line and the wild type leaves were randomized to eliminate any bias.

Transformation frequency of one event Nt6495-30 was approximately double that of the wild type control producing 7 versus 3 transformants respectively. Nt6495- 16 and Nt6495-69 had approximately the same transformation frequency (3 transformants) as the control. Thus, our preliminary analysis reveals that plastid transformation efficiency can have been enhanced by reducing the plastid number from wild type to less than 5 plastids per cell. Interestingly, all of the plastid transformant regenerants from Nt6495 lines were very much slower in growth and size compared to those from wild type. It appears that the presence of the selectable antibiotic spectinomycin dihydrochloride at a concentration of 500mg/ml can have affected the regenerability of cells in the Nt6495 lines. Thus, it is possible that there could be more plastid transformed cells in the transgenic Nt6495 lines which were susceptible to the antibiotic and could not regenerate. To check if this was the case, kill curves with lower concentrations of spectinomycin dihydrochloride (50, 100, 200, 300, 400 and 500mg/ml) can be used with each of the Nt6495-30, Nt6495-16 and Nt6495-69 lines to establish the concentration at which the regeneration of shoots are as good as in wild type. This concentration of spectinomycin dihydrochloride will then be used to repeat transformation frequency tests with the three Nt6495 lines.

To analyze for direct glyphosate selection, kill curves with varying levels of glyphosate will be established with the Nt6495 lines to find the best selection level. Plastid transformation vector pMON49218 will be used to bombard the Nt6495 lines and tested for direct selection using the optimized glyphosate level.

In Arabidopsis. FtsZl nuclear expression construct pCGN6495 was used to transform Columbia ecotype. Tl seeds were collected and about 100 kanamycin resistant seedlings were analyzed for alteration in plastid size and number following the same protocols as outlined for the tobacco section of this report. The transgenic plants were divided into three groups based on plastid number — I) 20 independent lines containing few large plastid (1-5), (II) 23 lines lines containing 5-20 plastids and (III) 50 lines containing wild-type plastid number were obtained. Selected T2 plants from each category were analyzed for number of transgene integration loci and sent to the growth chamber for T3 seed collection to identify homozygous plants. Such plants can be used in plastid transformations as described by Sikdar, et al. (1998) Plant Cell Reports, 18:20-24.

Transformed plants selected for aadA marker gene expression or glyphosate resistance are analyzed to determine whether the entire plastid content of the plant has been transformed (homoplasmic transformants). Typically, following two rounds of shoot formation and spectinomycin selection, approximately 50% of the transgenic plantlets which are analyzed are homoplasmic, as determined by Southern blot analysis of plastid DNA. Homoplasmic plantlets are selected for further cultivation.

Southern blot analysis is used to confirm the integration of the chimeric expression cassettes in the plastid genome. Preparation, electrophoresis, and transfer of DNA to filters is as described (Svab et al., (1993 supra)). Total plant cellular DNA can be prepared as described by Dellaporta et al. (1983) Plant Mol. Biol. Rep. 1: 19- 21). To visually observe the expression of marker genes such as GFP from the chloroplasts of transformed plants, various tissues are visualized utilizing a dissecting microscope. Protoplasts and chloroplasts are isolated as described in Sidorov. et al. (1994) Theor. Appl. Genet. 88:525-529. The above results demonstrate that the sequences of the present invention provide an efficient means for the production of plastid transformed plants. Furthermore, such methods find use in plastid transformation methods involving the selection of transplastomic plants on herbicides, for example glyphosate.

All publications and patent applications mentioned in this specification are indicative of the level of skill 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 claim.

Claims

CLAIMSWhat is claimed is:

1. In a method for transforming a plant cell plastid comprising the steps of introducing into cells of a plant a construct comprising a promoter functional in a plant cell plastid operably associated with a DNA sequence of interest and transforming said plant cell plastid with said construct, wherein the improvement comprises introducing said construct into a plant cell having an altered plant plastid morphology selected from the group consisting of altered plastid size and altered plastid number in said plant cell.

2. The method according to Claim 1 , wherein said plastid size is increased from a wild-type plant plastid morphology.

3. The method according to Claim 2. further wherein said plastid number is decreased from a wild-type plant plastid morphology .

4. The method according to Claim 1, wherein said plastid size is decreased from a wild-type plant plastid morphology.

5. The method according to Claim 4, further wherein said plastid number is increased from a wild-type plant plastid morphology.

6. The method according to Claim 2, wherein said plant cell is obtained from a plant tissue source in which plastid division is inhibited.

7. The method according to Claim 6, wherein said plastid division is inhibited by introduction into cells of the plant tissue source a second DNA construct comprising in the 5' to 3' direction of transcription a promoter functional in a plant cell, a DNA sequence coding for a gene involved in plastid cell division and a transcriptional termination sequence functional in a plant cell.

8. The method according to Claim 7, wherein said DNA sequence is in an antisense orientation.

9. The method according to Claim 8, wherein said construct contains a DNA sequence coding for an FtsZ protein.

10. The method according to Claim 7, wherein said DNA sequence is in a sense orientation.

1 1. The method according to Claim 10, wherein said DNA sequence provides for sense suppression.

12. The method according to Claim 6, wherein said plastid division is inhibited by growing a plant under culture conditions which inhibit the division of plant cell plastids.

13. The method according to Claim 12, wherein said culture conditions comprise growing the plant tissue source under exposure to an inhibitor of bacterial cell division.

14. The method according to Claim 13, wherein said inhibitor is 5,5'-Bis-(8- anilino- 1 -naphthalenesulfonate).

15. The method according to Claim 6, wherein said plastid division is inhibited by genetic mutagenesis.

16. An isolated DNA sequence encoding a plant FtsZ protein from Arabidopsis thaliana.

17. The DNA sequence of Claim 16, wherein said FtsZ protein is encoded by a sequence which includes a sequence selected from the group consisting of SEQ ID Nos:l and 3.

18. An isolated DNA sequence encoding a plant FtsZ protein from Brassica .

19. The DNA sequence of Claim 18, wherein said FtsZ protein is encoded by a sequence of SEQ ID NO:5.

20. An isolated DNA sequence encoding a plant FtsZ protein from soybean.

21. The DNA sequence of Claim 20, wherein said FtsZ protein is encoded by a sequence which includes a seqeunce selected from the group consisting of SEQ ID NOs:20-31.

22. An isolated DNA sequence encoding a plant FtsZ protein from corn.

23. The DNA encoding sequence of Claim 22, wherein said FtsZ protein is encoded by a sequence which includes a sequence selected from the group consisting of SEQ ID Nos: 10- 19.

24. A recombinant DNA construct comprising any of the DNA encoding sequences of Claims 16-23.

25. A plant cell comprising the DNA construct of 24.

26. A plant comprising a cell of Claim 25.

27. A method for improving the selectability of plant comprising, transforming a plant cell source having an altered plastid morphology with a construct comprising a promoter functional in a plant cell plastid operably associated with a nucleic acid sequence encoding a selectable marker.

28. The method according to Claim 27, wherein said nucleic acid sequence encodes an herbicide tolerance gene.

29. The method according to Claim 27, wherein said nucleic acid sequence encodes a glyphosate tolerance gene.

30. A method for preparing a plant cell source with increased plastid transformation efficiency comprising, transforming a plant cell with a construct comprising a promoter functional in plant cell operably associated with a nucleic acid sequence encoding a FtsZ protein.

31. A method for transforming a plant cell plastid comprising, introducing into a plant cell having altered plastid morphology a first nucleic acid construct comprising a promoter functional in a plant cell plastid operably associated with a nucleic acid sequence of interest.