# Patent

## Title
Plastid promoters for transgene expression in the plastids of higher plants

## Abstract

The present invention provides promoter elements useful for stably transforming the plastids of higher plants. The constructs described herein contain unique promoters that are transcribed by both nuclear encoded plastid RNA polymerases, plastid encoded plastid RNA polymerases or both. Use of the novel constructs of the invention facilitates transformation of a wider range of plant species and enables ubiquitous expression of a transforming DNA in plastids of multicellular plants.

## Background
The present application is a division of U.S. application Ser. No. 09/445,283, filed Dec. 3, 1999 now U.S. Pat. No. 6,624,296 which claims priority to International Application No. PCT/US98/11437, filed Jun. 3, 1998, which claims priority to U.S. Application No. 60/058,670, filed Sep. 12, 1997 and U.S. Application No. 60/048,376, filed Jun. 3, 1997, the entire disclosures of each being incorporated by reference herein.


Pursuant to 35 U.S.C. §202(c) it is acknowledged that the U.S. Government has certain rights in the invention described herein, which was made in part with funds from the National Science Foundation, Grant Number MCB96-30763.


FIELD OF THE INVENTION
The present invention relates to plant genetic engineering and particularly to plastid transformation in higher plants. The invention provides novel promoter sequences useful for the expression of foreign genes of interest in various plant species.
BACKGROUND OF THE INVENTION
Chloroplast genes are transcribed by an RNA polymerase containing plastid-encoded subunits homologous to the α, β and β′ subunits of E. coli RNA polymerase. The promoters utilized by this enzyme are similar to E. coli σ70-promoters consisting of −35 and −10 consensus elements (G. L. Igloi and H. Kossel, Crit. Rev. Plant Sci. 10, 525, 1992; W. Gruissem and J. C. Tonkyn, Crit. Rev. Plant. Sci. 12:-19, 1993) Promoter selection by the plastid-encoded RNA polymerase is dependent on nuclear-encoded sigma-like factors (Link et al. 1994, Plant promoters and transcription factors, Springer Verlag, Heidelberg, pp 63-83). In addition, transcription activity from some promoters is modulated by nuclear-encoded transcription factors interacting with elements upstream of the core promoter (L. A. Allison and P. Maliga, EMBO J., 14:3721-3730; R. Iratni, L. Baeza, A. Andreeva, R. Mache, S. Lerbs-Mache, Genes Dev. 8, 2928, 1994, Sun et al., Mol. Cell Biol. 9:5650-5659, 1989). These factors mediate nuclear control of plastid gene expression in response to developmental and environmental stimuli.
The existence of a second nuclear encoded polymerase transcription system in plastids has been demonstrated. However, the relevant nucleic acid sequences required for transcription initiation comprising the novel regulatory elements of this system have yet to be elucidated. It is an object of the present invention to provide these novel genetic elements. Incorporation of these regulatory elements into specific plastid directed DNA constructs enables greater flexibility and range in plant species available for plastid transformation, and facilitates ubiquitous expression of foreign proteins and/or RNAs and are useful in non-green plastids.
SUMMARY OF THE INVENTION
Promoters contain distinct DNA sequence information to facilitate recognition by the RNA polymerase and initiation of transcription leading to gene expression. In accordance with the present invention, promoters have been discovered which function in both monocots and dicots. These promoter elements may be used to advantage to express foreign genes of interest in a wider range of plant species. Additionally, the promoter elements of the invention drive expression of exogenous genes in non-green tissues. It is an object of the present invention to provide DNA constructs and methods for stably transforming plastids of multicellular plants containing such promoters. The DNA constructs of the invention extend the range of plant species that may be transformed.
The promoters recognized by plastid-encoded plastid RNA (PEP) polymerase have been well characterized in photosynthetic tissues such as leaf. The utility of PEP promoters for expression of foreign proteins in non-green tissues is demonstrated herein. The nuclear-encoded plastid (NEP) polymerase transcription system of the present invention directs expression of plastid genes also in roots, seeds, meristematic tissue and/or leaves. In most plants, including maize, cotton and wheat, plant regeneration is accomplished through somatic embryogenesis (i.e., involving meristematic tissue). In a preferred embodiment of the invention, efficient plastid transformation in these crops will be greatly facilitated, through the use of the NEP and PEP plastid transcription system and promoters of the present invention.
Particularly preferred promoters for use in the constructs of the invention are the clpP −111 (SEQ ID NOS: 15, 16, 30 and 31) promoters for the transformation of monocots and dicots and the pclp −53 promoter for transformation in dicots. Homologous clpP promoters from other plant species are contemplated to be within the scope of the present invention.
Other preferred promoters for use in expressing foreign genes of interest in the plant plastid in non-green tissues are PEP promoters present in the 16SrDNA operon, SEQ ID NOS: 28 and 29. Additional promoter elements suitable for use in the present invention are the rpoB and atpB promoters.
The NEP promoters of the invention are incorporated into currently available plastid transformation vectors such as those described in pending U.S. application Ser. No. 08/189,256, and also described by Svab & Maliga., Proc. Natl. Acad. Sci. USA, 90, 913 (1993). Protocols for using such vectors are described in U.S. Pat. No. 5,451,513. The disclosures of the three references cited above are all incorporated by reference herein. To obtain transgenic plants, plastids of non-photosynthetic tissues are transformed with selectable marker genes expressed from NEP promoters and transcribed by the nuclear-encoded polymerase. Likewise, to express foreign proteins of interest, expression cassettes are constructed for high level expression in non-photosynthetic tissue, using the NEP promoter transcribed by the nuclear-encoded plastid RNA polymerase. In another aspect of the invention, PEP promoters of the invention are incorporated into currently available plastid transformation vectors and protocols for use thereof.
In yet another aspect of the invention, the NEP transcription system also may be combined with the σ70-type system through the use of dual NEP/PEP promoters. In transforming DNA constructs, the promoters are arrayed in tandem, operably linked to the coding region of the foreign gene of interest. As used herein, the term transcription unit refers to isolated DNA segments which comprise the essential coding regions of one or more exogenous protein(s) of interest. Such transcription units may also contain other cis elements for enhancing gene expression, such as enhancer elements. Transcription units are operably linked to the promoters of the invention, such that expression of the transcription unit is regulated by said promoter. Particularly preferred promoters for use in combination are the Prrn PEP promoters combined with the clpP type II NEP promoter in dicots and the Prrn PEP promoter combined with the clpP Type I NEP promoter for use in both monocots and dicots. A suitable Prrn promoter has the following sequence (SEQ ID NO: 32) 5′-GCTCCCCCGC CGTCGTTCAA TGAGAATGGA TAAGAGGCTC GTGGGATTGA CGTGAGGGGG CAGGGATGGC TATATTCTG GGAGCGAACT CCGGGCGAAT ACGAAGCGCt TGGATACAGT TGTAGGGAGG GATT-3′.
Homologous PEP and NEP promoters from a variety of plant species corresponding to those listed above are also considered to fall within the scope of the present invention. The transforming DNA also contains 3′ regulatory regions of plant or bacterial origin to effect efficient termination of transcription. An exemplary 3′ regulatory region is shown in FIG. 13, SEQ ID NO: 27.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts a series of autoradiographs illustrating RNA steady state concentrations in green (G) and white (W) iojap maize leaves. To control for loading, blots shown above were reprobed for cytoplasmic 25S ribosomal RNA as described (Dempsey et al., Mol. Plant Path. 83:1021-1029, 1993) (lower panels).
FIGS. 2A and 2B depict a pair of autoradiographs showing the results of primer extension analysis on green (G) and white (W) iojap maize leaves for the mapping of the clpP promoter, FIG. 2A. The number −111 refers to the transcript 5′ end relative to the ATG translation initiation codon. FIG. 2B is an autoradiograph showing the results of an in vitro capping and RNase protection assay to identify primary transcript 5′ ends. Note that the RNase protection construct is short and protects only a 79 nt fragment. Size of molecular weight (MW) markers (100, 200, 300, 400, and 500 nucleotides) is also shown.
FIGS. 3A and 3B show the mapping of maize rpoB promoters. FIG. 3A is an autoradiograph showing the results of primer extension analysis of RNA from green (G) and white (W) iojap maize leaves. The number −147 refers to transcript 5′-end. FIG. 3B shows the results of an in vitro capping and RNase protection assay to identify primary transcript 5′-ends. Note that the RNase protection construct is short and protects only a 74 nt fragment. Size of molecular weight (MW) markers (100, 200, 300, 400, and 500 nucleotides) is also shown.
FIGS. 4A, 4B and 4C show the mapping of atpB promoters. FIG. 4A show the results of primer extension analysis of RNA from green (G) and white (W) iojap maize leaves. Number −298 and −601 refer to transcript 5′-ends. FIG. 4B shows the results of an in vitro capping and RNase protection assay to identify primary transcript 5′ ends. Note that the RNase protection construct is short and protects only a 79 nt fragment. Size of molecular weight (MW) markers (100, 200, 300, 400, and 500 nucleotides) is also shown. FIG. 4C depicts a physical map of the atpB-rbcL intergenic region. Map position of the primary transcript 5′ ends for NEP and PEP promoters are marked with filled and open circles, respectively.
FIG. 5A shows the alignment of DNA sequences flanking the NEP promoter transcription initiation sites. Site of transcription initiation is marked (filled circles). Regions with significant similarity are boxed (Box I and Box II). Sequences corresponding to the loose 10-nt dicot NEP consensus are underlined with thin lines. clpP −111 promoter region is maize is indicated by the thick underlining. FIG. 5A shows rpoB (SEQ ID NO: 1), atpB (SEQ ID NO: 2) and clpP (SEQ ID NO: 3) promoter regions, respectively in maize. The alignment and nucleotide sequences of atpB promoters in maize, sorghum, barley, wheat, and rice are shown in FIG. 5B and correspond to SEQ ID NOS: 4, 5, 6, 7 and 8 respectively. FIG. 5C shows the alignment of the rpoB sequences in maize (SEQ ID NO: 9), rice (SEQ ID NO: 10) and tobacco (SEQ ID NO:11). The alignment of the clpP sequences in maize (SEQ ID NO: 12), rice (SEQ ID NO: 13), and wheat (SEQ ID NO: 14 is shown in FIG. 5D.
FIG. 6 shows the sequence alignment of the tobacco (SEQ ID NO: 15) and rice (SEQ ID NO: 16) plastid clpP promoter regions. The NEP transcription initiation sites are marked with filled circles, the PEP transcription initiation site is marked with an open circle. The third tobacco NEP promoter is outside the sequence shown. The clpP coding regions are boxed. The 29-bp shared homologous region around the Type II tobacco PclpP −53 promoter is underlined.
FIG. 7 is a schematic drawing of the plastid targeting region of plasmid pDS44. On top is shown the restriction map of the chimeric uidA gene. PclpP is the rice clpP promoter fragment between the SacI and Nco site (SEQ ID NO: 17) which is shown at the bottom of FIG. 5. uidA encodes the β-glucuronidase reporter enzyme. Trps16 is the rps16 ribosomal protein gene 3′ untranslated region. A map of the transforming DNA in a pPRV111A plastid vector (Gene Bank Accession No. U12812) is also shown (Zoubenko et al., Nucleic Acids Res. 22:3819-3824, 1994).
FIGS. 8A and 8B illustrate the promoter activity of the rice clpP promoter region in transgenic tobacco plastids. FIG. 8A shows the results of primer extension analysis to map RNA 5′-ends upstream of the rice PclpP::uidA::Trps16 chimeric gene. Primer extension analysis was carried out on total cellular RNAs isolated from the leaves of wild-type (wt) and transplastomic (T) tobacco plants. The numbers (−61, −111, −136, −169, 177) refer to nucleotide positions of the mapped 5′ ends relative to the ATG translation initiation codon of the rice clpP gene. FIG. 8B shows a schematic representation of RNA 5′ ends mapped in the rice clpP promoter region in rice (Os) and in tobacco (Os in Nt), and upstream of the wild-type tobacco clpP gene (Nt). Promoters identified in homologous systems are marked (NEP, filled circles, PEP, open circles). Numbers indicate distance from translation initiation codon (nucleotide upstream of ATG is position −1).
FIG. 9 depicts an alignment of DNA sequences which are conserved around the plastid clpP transcription initiation sites. Sequences are aligned for Marchantia polymorpha (Kochi) (SEQ ID NO: 18), Pinus contorta (Clarke) (SEQ ID NO: 19), spinach (Westhof) (SEQ ID NO: 20), tobacco (Hajdukiewicz et al., 1977) (SEQ ID NO: 21), rice (SEQ ID NO: 22), maize (SEQ ID NO: 23) and Arabidopsis (SEQ ID NO: 24). Wild-type RNA 5′ ends are marked with filled circles. The −61 5′ end of the rice clpP-uidA chimeric mRNA is marked with an asterisk. Conserved nucleotides are boxed if identical at least in five species. The translation start codons are shaded.
FIG. 10 shows the plastid targeting region of plasmid pPS18. Plasmid pPS18 has the plastid targeting region of plastid vector pPRV111A, GenBank Accession No: U12812, with a chimeric uidA gene expressed from the PclpP −53 (−22/+25) promoter. DNA sequence of the uidA gene is shown in FIG. 13 (SEQ ID NO: 27).
FIG. 11 depicts the clpP 5′ fragments tested for promoter activity. The largest segment contains the PclpP −53 and PclpP −95 transcription initiation sites derived from Type II NEP and PEP promoters, respectively. The boundaries give the distance in nucleotides from PclpP −53 transcription initiation site (+1). SEQ ID NO: 25 contains the sequences present between −22 and +25 and SEQ ID NO: 26 shows the nucleotides present between −10 and +25. Both sequences function as promoters.
FIG. 12 depicts the results of primer extension analysis to test promoter activity of clpP 5′ fragments included in FIG. 11. Transcripts derived from PclpP −53 (−53), a minor NEP promoter (*), PclpP −95 (−95) and PclpP −173 (−173) are marked. DNA sequences on the side give distance from primer in nucleotides.
FIG. 13 shows the DNA sequence (SEQ ID NO: 27) of the chimeric uidA gene. SacI and HindIII cloning sites are marked. SacI, XhoI, NcoI, XbaI and HindIII sites are underlined. Translation initiation (ATG) and stop (TGA) codons are underlined twice. Nucleotides derived from the tobacco plastid genome are in capital letters; the position of the first and last nucleotide within the genome is listed.
FIG. 14 is a northern blot showing RNA steady state concentrations in leaf (L) and in embryogenic cultured cells (E) of rice. To control for loading, the blots were stripped and probed for cytoplasmic 25S ribosomal RNA (lower panels). Hybridization signals were quantified with a Molecular Dynamics PhosphorImager. The fold excesses in leaves over cultured cell signals are shown below the panels.
FIG. 15 is a Southern blot showing the relative plastid genome copy number in leaves (L) and in cultured embryogenic cells (E) of rice. EcoRI-digested total cellular DNA (approximately 2 μg per lane) was probed for plastid 16SrDNA (upper) and nuclear-encoded 25SrDNA (lower). Hybridization signals were quantified with a Molecular Dynamics PhosphorImager. The ratio of 16SrDNA/25SrDNA signal intensity in leaves relative to embryogenic cells was 1.1.
FIG. 16 is an autoradiograph depicting the mapping results of plastid mRNA promoters in rice leaves (L) and embryogenic cultured cells (E) using primer extension analysis. Numbers on the right indicate the distance between the translation initiation codon (ATG) and 5′ ends of primary transcripts (PEP, ◯; NEP, ●), or of processed mRNAs (−). DNA sequences on the left are size markers. Sequence ladders shown for 16S rDNA and clpP were obtained with homologous template and oligonucleotides used for primer extension analysis.
FIG. 17 shows the alignment of the maize (SEQ ID NO: 28 and rice (SEQ ID NO: 29) PEP promoter in the rrn operon, FIG. 17A. FIG. 17B shows the alignment of the plastid clpP promoter regions in maize (SEQ ID NO: 30) with the homologous regions in rice (SEQ ID NO: 31). PEP (◯) and NEP (●) transcription initiation sites and processed 5′ ends (−) are marked. For 16SrDNA sequence information see Strittmatter et al. (1985). The present invention provides sequence information for maize clpP.



DESCRIPTION OF THE INVENTION
Several reports have suggested the existence of an additional plastid-localized, nuclear-encoded RNA polymerase (reviewed in Gruissem and Tonkyn, 1993; Igloi and Kossel, 1992; Mullet, 1993; Link, 1994). By deleting the rpoB gene encoding the essential β subunit of the tobacco E. coli-like RNA polymerase, the existence of a second plastid transcription system which is encoded by the nucleus has been established (Allison et al., 1996, EMBO J. 15:2802-2809). Deletion of rpoB yielded photosynthetically defective, pigment-deficient plants.
While the activity of the previously-known plastid-encoded σ70-type transcription system in photosynthetically active tissues, such as leaf, has been the subject of much research, the nuclear-encoded polymerase transcription system has not yet been characterized. In accordance with the present invention, it has been discovered that the NEP system also directs expression in roots, seeds and meristematic tissue. In most plants, including maize, cotton and wheat, plant regeneration is accomplished through somatic embryogenesis (i.e., involving meristematic tissue). Efficient plastid transformation in these crops will be enabled, or greatly facilitated, through the use of clpP promoters driven by the NEP plastid transcription system of the present invention.
The ATP dependent Clp protease is widespread, if not ubiquitous, among both procaryotic and eucaryotic cells (Goldberg, Eur. J. Biochem. 203:9-23, 1992). Clp protease expression has recently been examined in chlamydomonas (Huang et al. Mol. Gen. Genetics 244:151-159, 1994). The results of gene knockout experiments in this unicellular algae demonstrated the following: 1) all the transformants were found to be heteroplasmic mutants containing both the disrupted clpP and wild-type copies; 2) the transformants persisted as heteroplasmic mutants after six rounds of growth and screening under selection pressure for the disrupted clpP; and 3) the heteroplasmic mutant stabilized at a level where approximately 80% of the clpP DNA copies were disrupted. These data indicate that the clpP is essential for cell growth even under conditions where photosynthesis is not required.
Shanklin et al. have examined the immunolocalization of clpP protein in Arabidopsis chloroplasts (Shanklin et al. The Plant Cell 7:1713-1722, 1995). These studies revealed that clpP and clpC are constitutively expressed in all tissues of Arabidopsis at levels equivalent to those of E. coli clpP and clpA. The observation that the clpP NEP promoter drives constitutive gene expression in all parts of a plant makes this promoter particularly suitable for use in the present invention.
The NEP promoters of the invention are incorporated into currently available plastid transformation vectors such as those described in pending U.S. application Ser. No. 08/189,256, and also described by Svab & Maliga., Proc. Natl. Acad. Sci. USA, 90, 913 (1993). Protocols for using such vectors are described in U.S. Pat. No. 5,451,513. The disclosures of the three references cited above are all incorporated by reference herein. To obtain transgenic plants, plastids of non-photosynthetic tissues as well as photosynthetic tissues are transformed with selectable marker genes expressed from NEP promoters and transcribed by the nuclear-encoded polymerase. Likewise, to produce foreign proteins of interest, expression cassettes are constructed for high level expression in non-photosynthetic tissue, using the clpP NEP promoter transcribed by the nuclear-encoded plastid RNA polymerase. The NEP transcription system also may be combined with the σ70-type system through the use of dual NEP/PEP promoters.
For versatility and universal applications, expression of selectable marker genes for plastid transformation is desirable in all targeted tissue types at a high level. Selectable marker genes in the currently utilized plastid transformation vectors are expressed from PEP promoters recognized by the plastid encoded RNA polymerase. The PEP polymerase transcribes photosynthetic genes and some of the housekeeping genes, and therefore appears to be the dominant RNA polymerase in photosynthetically active leaf tissues. Efficient plastid transformation has been achieved in tobacco based on chloroplast transformation in leaf cells. However, plant regeneration is not feasible, or is not practical from the leaves of most agronomically important cereal crops, including maize, rice, wheat and in cotton. In these crops, transgenic plants are typically obtained by transforming embryogenic tissue culture cells or seedling tissue. Given that these tissues are non-photosynthetic, expression of marker genes by constitutive clpP NEP promoters which are active in non-green tissues will facilitate transformation of plastids in all non-photosynthetic tissue types. Furthermore, as demonstrated herein, the ribosomal RNA operon PEP promoter is highly active in rice embryogenic cells.
The following nonlimiting Examples describe the invention in greater detail. Specifically, Examples I-III below describe preferred methods for making and using the DNA constructs of the present invention and for practicing the methods of the invention. Any molecular cloning and recombinant DNA techniques not specifically described are carried out by standard methods, as generally set forth, for example, in Ausubel (ed.), Current Protocols in Molecular Biology. John Wiley & Sons, Inc. (1994).
EXAMPLE I
Identification of Promoters for the Nuclear-encoded Plastid RNA Polymerase in the Ribosome-deficient Maize Mutant Iojap
As described previously, plastid promoters with conserved −10/−35 elements are well characterized for the plastid-encoded plastid RNA polymerase, PEP. Additionally, a ten-nucleotide promoter consensus was reported for a second, nuclear encoded, plastid RNA polymerase, NEP, in tobacco, a dicot (Hajdukeiwicz et al. 1997, in press). In this Example, NEP promoters active in monocots are described. NEP promoter mapping was carried out in the plastid ribosome-less maize mutant iojap which lacks PEP. These studies have revealed that atpB, an ATPase subunit gene, contains promoters for both NEP and PEP. In contrast, clpP, a protease subunit gene, and the rpoB operon, encoding the rpoB, rpoC1 and rpoC2 PEP subunit genes, are transcribed from NEP promoters only. These findings suggest conservation of transcription systems between monocots and dicots for the expression of certain plastid genes. The monocot NEP promoters share sequence homology around the transcription initiation site, including the 10-nucleotide loose consensus identified in dicots.
In the plastids of photosynthetic higher plants, genes are transcribed by at least two RNA polymerases: the plastid-encoded plastid RNA polymerase (PEP) and the nuclear-encoded plastid RNA polymerase (NEP). The sigma-factor homologues and PEP regulatory factors are encoded in the nucleus and are imported into plastids (Igoli and Kossel, (1992) supra; Link, (1996) Bioessays 18:465-471; Stern D B, Higgs D C, Yang J (1997) Trends Plant Sci 2:308-315). Much less is known about NEP. One appealing candidate for NEP is a 110 kD protein which has biochemical properties similar to yeast mitochondrial and T7 RNA polymerases. NEP may require accessory factors, such as CDF2 for its activity (Lerbs-Mache, 1993; Iratni et al., 94; Genes and dev).
Dicot promoters for NEP were identified in tobacco plants lacking PEP due to deletion of rpoB encoding the PEP β subunit. The general rule emerging from these studies is that plastid genes fall into three classes. Class I genes contain only PEP promoters. Examples of this class are the photosytem I and II genes. Class II genes contain both NEP and PEP promoters. Representative members of this class of genes are genes involved in plant metabolism and housekeeping genes. Genes in the third class contain NEP promoters only. Genes in this class include rpoB and accD.
As plastid transformation is not yet available in monocots, ΔrpoB plants could not be obtained for maize NEP promoter analysis. However, mutants with a defect in plastid ribosome accumulation are available in barely (albostrians; Hess et al., EMBO J. 12:563-571, 1993) and maize (iojap; Walbot and Coe, Proc. Natl. Acad. Sci. 76:2760-2764, 1979; Han et al., Planta 191:552-563, 1993; Han et al., EMBO J. 11:4037-4046, 1992). In the absence of plastid ribosomes, these mutants cannot synthesize PEP. Both mutants accumulate mRNAs for a subset of plastid genes indicating the presence of NEP activity (Hess et al., 1993, supra; Han et al., 1993, supra).
NEP promoters for three plastid genes were mapped in white ribosome-less maize iojap seedlings. The data show that the maize atpB gene has alternative NEP and PEP promoters while the clpP and the rpoB genes are transcribed from NEP promoters exclusively in both white and green seedlings. DNA sequence alignment revealed that monocot NEP promoters share homology directly upstream of the transcription initiation site. The homologous region includes the previously identified tobacco NEP promoter consensus elements suggesting conservation of the NEP transcription machinery between monocots and dicots.
Materials and Methods for Example I
Plant Materials. Iojap is a recessive striped mutant of maize. Maternal white and green seedlings were obtained by crossing a striped ij/ij maternal parent (1404) with pollen from a wild type male (inbreed Oh51a). The seeds were kindly provided by Rob Martienssen and Mary Byrne, Cold Spring Harbor Laboratory. Surface-sterilized seeds were germinated in vitro on 2% MS medium (24° C., 16 hours illumination).
RNA Gel blots. Total cellular RNA was prepared from the leaves of 9-day-old seedlings (Stikema et al. Plant Mol. Biol. 11:255-269, 1988). The RNA (5 μg per lane) was electrophoresed on 1% agarose/formaldehyde gels, then transferred to Hybond N (Amersham) using the Posiblot Transfer apparatus (Stratagene). Hybridization to random-primer labeled fragment was carried out in Rapid Hybridization Buffer (Amersham) overnight at 65° C.
Double-stranded ptDNA probes were prepared by random-primed 32P-labeling of PCR-generated or gel-purified DNA fragments. The sequence of the primers used for PCR, along with their positions within the tobacco (N.t.; Genebank Accession No. Z00044) or maize (Z.m.; Genebank Accession No. X86563) ptDNA are as follows:














5′ nt

SEQ



position

ID


Gene
in ptDNA
Sequence
NO:



















atpB
(Z.m.)
55860 (C)
GAGAGGAATGGAAGTGATTGACA
(33)




55103
GAGCAGGGTCGGTCAAATC
(34)


clpP
(Z.m.)
69840
ATCCTAGCGTGAGGGAATGCTA
(35)




70064 (C)
AGGTCTGATGGTATATCTCAGTAT
(36)


psbA
(N.t.)
 1550 (C)
CGCTTCTGTAACTGG
(37)




 667
TGACTGTCAACTACAG
(38)









The following ptDNA fragments were used as probes: rbcL (N.t.), a BamHI fragment (nucleotides 58047 to 59285 in ptDNA); 16SrDNA (N.t.), EcoRI to EcoRV fragment (nucleotides 138447 to 140855 in the ptDNA); rpoB, HindIII fragment (nt 24291-24816).
Primer extension analysis. Primer extension reactions were carried out on 10 μg of total leaf RNA as described (Allison et al. EMBO J. 15:2802-2809, 1996). The primers are listed below, with nucleotide positions in the published maize plastid genome sequence (Maier et al., J. Mol. Biol. 251:614-628, 1995). Underlined oligonucleotides (added to create cloning site) were also used to generate the capping constructs, in which case the position of the first nucleotide in the genome sequence is positioned immediately following the underline.














5′ nt

SEQ



position

ID


Gene
in PtDNA
Sequence
NO:







clpP#1
70182
GGTACTTTTGGAACACCAATGGGCAT
(39)


atpB#1
56095
GAAGTAGTAGGATTGGTTCTCATAAT
(40)


atpB#2
56640
GGTCTAGAATTCCTATCGAATTCCTTC
(41)


rpoB#1
21545 (C)
GAATCTACAAAATCCCTCGAATTG
(42)


rpoB#2
21418 (C)
ACTCTTCATCAATCCCTACG
(43)









(Note: C at 3′-end of the atpB#2 oligonucleotide is at nt position 56644; the published sequence has a 15 nucleotide deletion relative to the sequence we found).

Identification of primary transcripts by in vitro capping. Total leaf RNA (20 μg for clpP or 100 μg for rpoB and atpB transcripts) from white seedlings was capped in the presence of 0.25 or 1.0 mCi [α-32p] GTP (Kennell and Pring, Mol. Gen. Genet. 216:16-24, 1989). Labeled RNAs were detected by ribonuclease protection (Vera et al., Plant Mol. Biol. 19:309-311, 1992) using the RPAII kit (Ambion). To prepare the protecting complementary RNA, an appropriate segment of the plastid genome was PCR-amplified using the primers listed below.














5′ nt

SEQ



position

ID


Gene
in PtDNA
Sequence
NO:



















clpP#2
70241
GGTCTAGACTACACTTTAATATGGA
(44)



clpP#3
70549 (C)
GGGAATTCTGTTTGTAAGAAGA
(45)


atpB#2
56640
GGTCTAGAATTCCTATCGAATTCCTTC
(41)


atpB#3
56832 (C)
GGCTCGAGGGACAACTCGATAGGATTAGG
(46)


rpoB#3
21394 (C)
GGTCTAGAATCTAGCAATCATGGAATC
(47)


rpoB#4
21066
GGCTCGAGCGTGCTATTCTAAATCGT
(48)









The 5′ primers set forth above were designed to add a Xba I restriction site (underlined) upstream of the amplified fragment. The 3′ primers were designed to add a XhoI (atpB , rpoB) or EcoRI (clpP) site (underlined) downstream of the amplified sequence. The amplified product was cloned after digestion with the appropriate restriction enzyme into a pBSKS+ vector (Stratagene). To generate unlabeled RNA complementary to the 5′ end of RNAs, the resulting plasmid was linearized with XhoI (atpB, rpoB) or EcoRI (clpP) and transcribed in a Megascript (Ambion) reaction with T7 RNA polymerase. Markers (100, 200, 300, 400, and 500 nucleotides) were prepared with the RNA Century Markers Template Set (Ambion), following the manufacturer's protocol.
DNA sequence analysis. DNA sequence analysis was carried out utilizing the Wisconsin Sequence Analysis Package (Genetics Computer Group, Inc.).
Results and Discussion
Plastid Transcript Accumulation in the Maternal White and Green Maize Plants.
Lack of 16S rRNA accumulation in the white maize plants confirmed the reported lack of plastid ribosomes in the white iojap seedlings (Walbot and Coe 1979, supra). The absence of rbcL and psbA mRNAs, known to be transcribed from PEP promoters, indicates that the maternal white plants indeed lack PEP activity. (Han et al., 1993, supra). See FIG. 1.
Transcript accumulation for three additional plastid genes: clpP, rpoB and atpB has been analyzed in iojap plants. High steady-state mRNA levels, indicating the presence of NEP promoters was reported for these genes in iojap maize (Han et al., 1993, supra), albostrians barley (Hess et al., 1993, supra) and ΔrpoB tobacco (Hajdukiewicz et al., EMBO J., in press, 1997), respectively. Readily-detectable accumulation of mRNA in the ribosome-less iojap plants confirmed that active NEP promoters regulate expression for each of these genes (FIG. 1).
The clpP NEP Promoter is Efficiently Transcribed in White and Green Seedlings.
To identify the NEP promoters, transcript 5′-ends were mapped by primer extension analysis. To distinguish between 5′-ends that represent transcripts from NEP promoters from those generated by RNA processing, the 5′-ends were capped using guanylyltransferase.
For clpP, significant mRNA accumulation was found in both white and green seedlings. Primer extension analysis identified only one major clpP 5′-end at nucleotide position −111 (the nucleotide upstream of the ATG being at −1 position). The clpP −111 transcript could be capped in vitro. See FIG. 2B. These data confirm that this 5′ end is a primary transcript and also identifies the maize NEP promoter PclpP −111. The same 5′-end is observed in both white and green maize seedlings indicating that the same clpP promoter is active in chloroplasts as well as in the nonphotosynthetic iojap plastids (FIG. 2A). Therefore, PclpP −111 is considered to be a constitutive promoter. In rice, the clpP transcript 5′-end mapped to the same nucleotide indicating conservation of PclpP −111 in monocots (data not shown).
The rpoB NEP Promoter Activity is Enhanced in iojap Plastids.
RNA gel blot analysis shows that rpoB mRNA accumulates to a detectable level only in white seedlings (FIG. 1). However, more sensitive primer extension analysis revealed that the same 5′-ends were present in both white and green leaves. Two 5′-ends could be identified, a major band at nucleotide position -147, and a minor band at position −285 (FIG. 3A). The in vitro capping assay confirmed that the −147 RNA species is a primary transcript (FIG. 3B), and therefore the product of PrpoB −147 promoter.
The atpB Gene is Transcribed From a NEP Promoter in White Plants and from a PEP Promoter in Green Seedlings.
There is substantial atpB mRNA accumulation in green leaves, while much less is found in the white iojap leaves (FIG. 1). Primer extension analysis of mRNA from green leaves identified a transcript with a 5′-end at nucleotide position −298 (FIG. 4A) confirming an earlier report (Mullet et al., Plant Mol. Biol. 4:39-54, 1985). This −298 mRNA species was absent in leaf RNA isolated from white plants, indicating that the −298 mRNA is a PEP transcript. Instead, another atpB transcript was mapped to nucleotide position −601 (FIG. 4A). The difference in the size of the two mRNAs is apparent on the RNA gel blot shown in FIG. 1.
The −601 transcript could be capped in vitro by guanylyltransferase, indicating that it is a primary transcript (FIG. 4B). Therefore, it is the product of the PatpB −601 NEP promoter, with readily detectable activity only in white iojap leaves.
Sequence Conservation Around the Monocot NEP Transcription Initiation Sites.
Alignment of the maize clpP, rpoB and atpB NEP promoters revealed significant homology upstream of the transcription initiation sites. In a 13-nucleotide region 8 nucleotides are shared in all three promoters (FIG. 5; Box I). In a pairwise comparison, atpB/clpP, atpB/rpoB and clpP/rpoB share 11, 10 and 8 nucleotides, respectively. In addition, atpB and clpP share 8 out of 9 nucleotides further upstream (−21 to −30 relative to transcription initiation site; Box II in FIG. 5).
Interestingly, each maize NEP promoter has sequence similarity around the transcription initiation site with the loose dicot NEP promoter consensus CATAGAATA/GAA (SEQ ID NO:63) (Hajdukiewicz et al., 1997, supra; underlined in FIG. 5). For clpP, 9 nucleotides are conserved out of 10; for atpB and rpoB, the number of conserved nucleotides is 7 out of 10 (FIG. 5). In addition, a second conserved region (Box II, FIG. 5) is found upstream of Box I in atpB and clpP, but not in the rpoB promoters. Interestingly, the moncot Box II contains truncated versions of the dicot NEP consensus in a direct orientation: 7 out of 10 bp match in case of the maize clpP (ATAGAAT) and atpB (AT-GAATA) genes (FIG. 5). These tandem repeats may play a role in regulating NEP promoter activity.
The regions containing the tentative maize NEP promoter sequences (−30 to +25) have been aligned with homologous regions from other monocot plants. See FIG. 5B-5D. The high degree of sequence conservation indicates the presence of functional promoters in each of these monocots species. Promoter activity for the rice homologue of the maize PclpP −111 promoter has been confirmed by primer extension analysis (data not shown).
The data reported herein show that maize plastid NEP promoter regions share sequence homology around the transcription initiation site with the conserved CATAGAATA/GAA (SEQ ID NO:63) NEP sequence motif in tobacco (FIG. 5). Therefore, these promoters are considered to be Type I NEP promoters. This finding indicates conservation of the NEP transcription machinery between monocots and dicots. Sequences upstream of the transcription initiation sites are conserved more extensively than downstream sequences among the maize clpP, rpoB, and atpB promoters, as shown in FIG. 5, similar to the dicot Type I NEP promoters (Hajdukiewicz et al., 1997, supra).
Both, the maize PclpP −111 promoter and the tobacco PclpP −53 promoter are constitutive. In contrast to the maize promoter, the clpP promoter region in tobacco lacks the CATAGAATA/GAA (SEQ ID NO:63) sequence motif (Hajdukiewicz et al., 1997, supra) suggesting recognition by a different NEP specificity factor (Type II NEP promoter). Interestingly, in Type II NEP promoters sequences downstream of the transcription initiation sites are conserved more extensively than upstream as described in the following example. The tobacco PclpP −53 promoter homologues are the only known examples for plastid Type II NEP promoters. They have been highly conserved during evolution, including the liverworth Machantia polymorpha and the conifer Pinus contorta. Although DNA sequences required for clpP Type II NEP promoter function are maintained, this region is transcriptionally silent in maize, rice and wheat. Lack of transcription from this region in cereals is probably due to the loss of Type II recognition specificity. See Example II. Accordingly, the tobacco (dicot) Type II clpP promoter is suitable to drive the expression of plastid transgenes only in dicots, whereas the cereal Type I promoter may be useful in both monocots and dicots.
The rpoB operon is one of few genes known to be expressed by NEP only. The PrpoB −147 is a Type I NEP promoter but, unlike clpP and atpB, lacks Box II (FIG. 5). Accumulation of mRNA from this promoter is low in mature leaves due to down-regulation of transcription rates (Baumgartner et al., Plant Physiology 101:781-791, 1993). The PrpoB -147 promoter identified in this study probably plays a central role in plastid development since it regulates expression of four out of the five plastid-encoded PEP subunits (Shimada et al., Mol. Gen. Genet. 221:395-402, 1990). According to one model, the two RNA polymerases form a developmental cascade during chloroplast differentiation. During the early stages of plastid development, plastid genes encoding the plastid's transcription and translation apparatus would be transcribed NEP. Once PEP is made, it would initiate transcription of photosynthetic genes from PEP promoters, and take over transcription of housekeeping genes from alternative PEP promoters (Hess et al., 1993, supra; Lerbs-Mache, Proc. Natl. Acad. Sci. 90:5509-5513, 1993; Mullet, Plant Physiol. 103:309-313, 1993; Hajdukiewicz et al. 1997, supra). Consistent with this model is transcription of rpoB from a NEP promoter. However, in maize at least one gene, clpP, is exclusively and efficiently transcribed by NEP in mature chloroplasts indicating that NEP remains active and essential for cellular functions even if PEP is present. An alternative hypothesis is proposed herein, which assumes that NEP and PEP are constitutively present all the time and selective transcription is mediated by promoter-specific transcription factors. The identification of NEP promoters for dicots (Hajdukiewicz et al., 1997, supra) and monocots (described herein) facilitates the elucidation of the roles these two plastid RNA polymerases play in plastid function and development.
EXAMPLE II
Altered clpP Promoter Recognition by the Nucleus-Encoded Plastid RNA Polymerase Suggests Loss of a Conserved Plastid Transcription Factor in Monocots
The plastid clpP gene is transcribed by the nuclear-encoded plastid RNA polymerase (NEP) in rice, a monocot, and tobacco, a dicot. However, the two NEP promoters do not share sequence homology. To assess conservation of NEP promoter recognition between monocots and dicots, a reporter gene (uidA) expressed from the rice clpP promoter region has been introduced into tobacco plastids. The data indicate that in tobacco, transcription initiates at the correct site from the rice clpP promoter. Thus, NEP promoter recognition for this gene is conserved in both monocots and dicots. Surprisingly, transcription from the rice sequence initiated at a second site, which possesses a short stretch of homologous sequence similar to the tobacco clpP promoter region. Sequences around the clpP transcription initiation site are conserved in tobacco, a dicot, Marchantia polymorpha, a bryophyte, and Pinus contorta, a conifer. Lack of transcription from this region in rice and other cereals indicates the evolutionary loss of a factor required for NEP Class II promoter specificity.
Materials and Methods for Example II
Construction of plasmid pDS44.
Plasmid pDS44 is a pLAA24 derivative (Zoubenko et al., Nucleic Acids Res. 22:3819-3824, 1994) which carries a uidA reporter gene expressed from a Prrn promoter. Plasmid pDS44 was obtained by excising the Prrn promoter as an SacI/EcoRI fragment and replacing it with the rice clpP promoter region engineered as a SacI/EcoRI fragment. The 251 nucleotide SacI/EcoRI DNA fragment containing the rice clpP promoter region (including 19 basepairs of the coding region) was obtained by PCR amplification. The sequence of the PCR primers, and the position of their first nucleotide (or of its complement) in the rice plastid genome (Hiratsuka et al., Mol. Gen. Genet. 217:185-194, 1989; GeneBank Accession No. X15901) are:










P1





68520 (C)
gggagcTCGAATCACCATTCTTT
SEQ ID NO: 49


 


P2


68270
gggaattcTTGGAACACCAATGGGCAT
SEQ ID NO: 50






Nucleotides derived from the plastid genome are in capital letters; those included to create a restriction site are in lower case letters. SacI or EcoRI restriction sites are underlined.

Tobacco plastid transformation. Plastid transformation and regeneration of transgenic tobacco plants was carried out according to the protocol described by Svab and Maliga (Proc. Natl. Acad. Sci. 90:913-917, 1993). Briefly, tobacco leaves were bombarded with tungsten particles coated with plasmid pDS44 DNA using the DuPont PDS 1000He Biolistic gun. Transgenic shoots were selected on RMOP medium containing 500 μg/ml spectinomycin dihydrocloride. Putative primary transformants were identified by histochemical staining for β-glucuronidase activity encoded by uidA (Jefferson, In Genetic Engineering, Vol. 10, Settlow, J. K., ed. Plenum Press, NY & London, pp 247-263, 1988). A uniform population of transformed plastid genomes was verified by Southern analysis (data not shown).
Primer extension analysis to map RNA 5′-ends.
Total leaf RNA was isolated from leaves of in vitro grown plants by the method of Stiekema et al., supra. Primer extension reactions were carried out on 20 μg of RNA with primer uidA PE1 as described by Allison and Maliga (1995) using primer P3: 5′-GGCCGTCGAGTTTT TTGATTTCACGGGTTGGGG-3′ (SEQ ID NO:51) (which is complementary to the uidA coding region.
DNA Sequence Analysis. DNA sequence analysis was carried out utilizing the Wisconsin Sequence Analysis Package (Genetics Computer Group, Inc.) as described in Example IV.
Results and Discussion
Construction of Transgenic Plants. The sequence of rice clpP upstream region included as a promoter fragment in plasmid pDS44 is shown in FIG. 6. In rice, this region contains the Os-PclpP −111 Type I NEP promoter. The cognate sequence in tobacco contains two NEP promoters and one PEP promoter. The Os-PclpP −111 NEP promoter was cloned upstream of a uidA coding region (encoding β-glucuronidase or GUS) with a ribosome binding site, and the rps16 3′-untranslated region (Trps16) for stabilization of the mRNA shown in FIG. 7. The chimeric uidA gene was cloned next to a selectable spectinomcyin resistance (aadA) gene in the pPRV111A plastid vector, Genebank Accession No. U12812, and introduced into tobacco plastids. A schematic drawing of the vector is shown in FIG. 7. Plastid transformants were selected on spectinomycin medium. Out of 25 spectinomcyin resistant lines, 12 were GUS positive. Integration of uidA at the target site was confirmed by Southern analysis (data not shown).
Primer extension analysis to test promoter activity. Primer extension analysis was carried out to map uidA 5′-ends initiating in the Os-PclpP −111 promoter region. Three major and two minor uidA transcripts were identified in the leaves of transgenic plants (FIG. 8A). A major transcript 5′ end mapped to nucleotide position −111, the same position as in rice. This result indicates that the rice Type I PclpP −111 promoter is properly recognized in tobacco, a dicot, indicating the broad applicability of this promoter in a variety of plant species. The second major transcript with the most intense signal was found at the rice −61 position. This transcript 5′-end mapped to a rice sequence with a 29 nucleotide stretch of homology to the tobacco Nt-PclpP −53 promoter region (FIG. 6). This was unexpected, since this region in rice and maize is transcriptionally silent (Example I). The third major transcript mapped to position −136 which falls within the upstream monocot NEP box (Example I).
The two minor transcript 5′-ends mapped to nucleotide positions −169 and −177 (FIG. 8). These transcripts are absent in rice leaf RNA, and they do not correspond to any of the known tobacco clpP transcripts. Therefore, these 5′-ends may be primary transcripts of fortuitous PEP or NEP promoters, or are processed mRNA 5′-ends.
Sequence alignment of the regions containing the clpP promoters The rice clpP promoter fragment was transcriptionally active in tobacco. This promoter sequence corresponds to the tobacco Type II PclpP −53 promoter. In this region, rice and tobacco share a 29 nucleotide stretch of homologous sequence with 23 conserved nucleotides. To test the conservation of this same region during evolution, sequences around the clpP transcription initiation sites were aligned, including those of the liverwort Marchantia polymorpha (Kohchi et al., Curr. Genet. 14:147-154, 1988), the conifer Pinus contorta, (Clarke et al., Plant Mol. Biol. 26:851-862, 1994), dicots tobacco (Hajdukiewicz et al., 1997, supra) spinach, Arabidopsis (Westhoff, Mol. Gen. Genet. 201:115-123, 1985) and the monocots maize and rice. See FIG. 9. With the exception of tobacco, a single transcription initiation site was mapped for each of the clpP genes. We have found, that the 29-nucleotide segment around the clpP transcription initiation site is conserved, (underlining) suggesting that clpP is transcribed by NEP in all of these species.
The data presented in this Example show that the rice Os-PclpP −111 NEP promoter is properly recognized in tobacco plastids. Transcription from the rice PclpP −111 NEP promoter in tobacco was observed presumably because the region around the transcription initiation site includes the dicot Type I NEP promoter consensus. As in rice, the Os-PclpP −111 NEP promoter is active in mature tobacco leaves. Therefore, it belongs to the relatively small number of Type I NEP promoters which are active in mature chloroplasts as well as proplastids.
Transcription from the rice clpP 5′ region at a second site, with a short stretch of homologous sequence to the tobacco PclpP −53 Type II NEP promoter was unexpected. Since rice contains all the cis elements required for Type II promoter activity, lack of transcription in rice should be due to the evolutionary loss of the specificity factor required for Type II promoter recognition. This specificity factor is well conserved during evolution, as evidenced by an active Type II clpP promoter in the liverwort Marchantia polymorpha and the conifer Pinus contorta (FIG. 9).
Experiments reported here for the rice Os-PclpP −111 NEP promoter suggest that the Type I NEP transcription machinery is sufficiently conserved between monocots and dicots to ensure faithful recognition of heterologous promoters. The clpP mRNA accumulates to significant levels in all plastid types (Shanklin et al., 1995, supra; Hajdukiewicz et al., 1997, supra; Example I). Therefore, the strong, constitutive Os-PclpP −111 NEP promoter is suitable for the expression of chimeric genes in a broad range of crops.
EXAMPLE III
In vivo Definition of a Type II Promoter, PclpP −53, for the Nuclear Encoded Plastid RNA Polymerase (NEP)
In tobacco the clpP gene is transcribed from three major NEP promoters initiating transcription −511, −173 and −53 nucleotides upstream of the translation initiation codon, and from a PEP promoter (5′ end at -95). Transcription from the Type II PclpP −53 NEP promoter is maintained in the green leaves of wild-type tobacco plants. Therefore, given its potential to drive the expression of selectable marker genes, the constitutive PclpP −53 promoter was chosen for analysis.
Materials and Methods for Example III
Construction of Plasmids. Plasmid pPS8 contains a uidA reporter gene as a SacI-HindIII fragment in a pBSKS+ plasmid (Stratagene). The chimeric uidA gene consists of: Between the SacI and XhoI sites, the PclpP −53 (−22/+25) promoter fragment containing 22 nt upstream and 25 nt downstream (+1 is nt where transcription initiates) of the clpP transcription initiation site; Between XhoI and NcoI sites, a ribosome binding site; Between the NcoI and XbaI sites, the uidA coding region with an N-terminal c-myc tag corresponding to amino acids 410-419 (EQKLISEEDL; SEQ ID NO: 52) within the carboxy terminal domain of the human c-myc protein (Kolodziej and Young, Meth. Enz. 194:508-519, 1991); Between the XbaI and HindIII sites the 3′ untranslated region of the rps16 ribosomal protein gene (Trps16). DNA sequence of the chimeric uidA gene between the SacI and HindIII sites in plasmid pPS8 is shown in FIG. 13. Relevant restriction sites of the chimeric uidA gene are shown in FIG. 10, where the uidA gene is shown as part of plasmid pPS18. Plasmid pPS18 was obtained by cloning the uidA gene as a SacI-HindIII fragment into SacI-HindIII-digested pPRV111A plastid transformation vector. Plasmid pPRV111A, a pBSKS+ plasmid derivative (Strategene), and was described in Zoubenko et al., 1994, supra.
Plasmids pPS16, pPS37, pPS17 and pPS38 listed in FIG. 11 were obtained from plasmid pPS18 by replacing the PclpP −53 (−22/+25) SacI-XhoI promoter fragment with the PclpP −53 (−152/+154), PclpP −53 (−152/+41), PclpP −53 (−152/+10), PclpP −53 (−39/+154) promoters, respectively. The SacI-XhoI fragments were obtained by PCR amplification. PCR primers are listed according to the position of the terminal nucleotide relative to the transcription initiation site (it is the complement of nt 74557 in the tobacco plastid genome: accession no. Z00044):










clpP−152
ccgagctcGAATGAGTCCATACTTAT
SEQ ID NO: 53



 


clpP−39
ccgagctcAAAACCAATATGAATATTATA
SEQ ID NO: 54


 


clpP−22
ccgagctcTATAAAGACAATAAAAAAAAT
SEQ ID NO: 55


 


clpP+10
ccctcgaGAAACGTAACAATTTTTTTT
SEQ ID NO: 56


 


clpP+25
ccctcgagTTTCACTTTGAGGTGGA
SEQ ID NO: 57


 


clpP+41
ccctcgagAGAACTAAATACTATATTTC
SEQ ID NO: 58


 


clpP+154
ccctcgagATATGACCCAATATATCTG
SEQ ID NO: 59






Anchor sequences derived from the plastid genome are in capital letters; added nucleotides to create restriction sites (underlined) are in lower case letters.
Tobacco Plastid Transformation. Plastid transformation and regeneration of tobacco plants was carried out as described by Svab and Maliga, 1993, supra. Transgenic plants were selected on regeneration medium containing 500 μg/ml spectinomycin dihydrochloride.
Primer Extension Analysis. Total leaf RNA was isolated from the leaves of transgenic plants maintained on RM medium, by the method of Stiekema et al.,1988, supra. Primer extension reactions were carried out as described by Allison and Maliga, 1995, supra, using 15 μg of the total RNA and primer PE1 complimentary to the 5′ end of the uidA coding sequence.

    Primer PE1 sequence:
    5′-GGCCGTCGAGTTTTTTGATTTCACGGGTTGGGG-3′ (SEQ ID NO: 51)

Results and Discussion



To identify functionally important sequences in the Type II PclpP −53 promoter, expression of reporter genes driven by sequences surrounding the clpP −53 NEP transcription initiation site was measured. Deletion of sequences from the 5′ and 3′ ends facilitated the determination of the boundaries of the promoter. These studies revealed that a 47-bp fragment is sufficient to support accurate transcription initiation. The data further suggest that not more than 28 bp out of 47 are essential for promoter function. A majority of the relevant sequences are downstream of the transcription initiation site.
Since all plastid promoters are within about 150 nucleotides of the transcription initiation site, transcription initiation in vivo from a 306-bp fragment (−152/+154) surrounding the clpP −53 transcription initiation site was assessed. For testing promoter function, clpP fragments were cloned upstream of a uidA reporter construct encoding D-glucuronidase. See FIG. 11. The uidA construct has a ribosome binding site between the XhoI and NcoI restriction sites, as well as the 3′-untranslated region of the plastid rps16 gene (Trps16) for stabilization of the mRNA. The chimeric uidA gene was introduced into tobacco plastids by linkage to a selectable spectinomycin resistance (aadA) gene. The 306-bp fragment contains a PEP and a NEP promoter (FIG. 11). Functioning of both promoters was established by primer extension analysis. See FIG. 12.
After confirming that the 306-bp fragment is sufficient to drive NEP transcription, a series of deletions were made from the 5′ as well as 3′ end. These constructs were then tested for transcription initiation in vivo in tobacco. The schematic design of the promoter deletions is shown in FIG. 11, the primer extension data are shown in FIG. 12. Primer extension analysis on the series of deletions showed that sequences extending from −10 to +25 (pPS43; FIG. 12) and −5 to +25 (pPS44; not shown) are sufficient to support accurate initiation from clpP −53. Upon overexposure, a faint band or the proper size is observed even in the +1 to +25 construct (pPS45, not shown). Also, transcription from the NEP promoter was abolished completely in the −152/+10 construct, (pPS17; FIG. 12) and in the −22 to +21 construct, (pPS41; not shown). This indicates that sequences between +5 and +25 are important for transcription from the NEP promoter. Some of this data is summarized in FIGS. 11 and 12.
Sequences required for transcription by the NEP polymerase are not known. Based on conservation of the ATAGAATA/GAA (SEQ ID NO:64) around the transcription initiation site, Hajdukiewicz et al., 1997, supra, most NEP promoters were classified as Type I. The promoter studied here, PclpP −53, lacks this sequence motif, because of which it is classified as Type II. Transcription analysis in the truncated promoter fragment of plasmid pPS18 in vivo shows that sequences required to support transcription from PclpP −53 are located within a 30 basepair fragment extending from −5 to +25 with respect to the transcription initiation site. Furthermore, nucleotides between +10 and +25 are important for transcription initiation, since there is no transcription from clpP promoter derivatives in plasmid pPS17 and pPS41 lacking this region.
Expression of the rice clpP promoter region revealed a transcript that mapped to a region with homology to the transcription initiation site of the PclpP 53 promoter. Alignment of the rice and the tobacco sequences show significant homology downstream of the transcription initiation site, and not much homology upstream. Considering that this rice sequence was recognized by tobacco in vivo (Example II), sequences that are important for transcription initiation should be present in the rice sequence. Combining this information with the finding that 22/+25 sequences are sufficient for transcription initiation in vivo, it appears that sequences 5′-ATTGTTACGTTTCCACCTCAAAGTGAAA-3′ (nucleotides 36 to 53 of SEQ ID NO: 25) extending from 3 to +25 contain the information which is important for PclpP 53 promoter function.
Since the constitutive Type II PclpP −53 is efficiently transcribed in all tissue types, it is useful for the expression of selectable marker genes, and of proteins of economic value in all dicot plants.
EXAMPLE IV
Plastid Promoter Utilization in Rice Embryogenic Cell Culture
The utilization of the clpP promoter in tobacco and maize plastids is described in the previous examples. The present example is directed to the analysis of plastid promoter utilization in rice. The 5′ ends of several mRNA species were mapped in samples derived from cultured embryogenic rice cells and leaves. The RNAs for clpP and 16SrRNA are relatively abundant in embryogenic rice cells indicating that the promoters of these genes may be used to advantage to drive plastid expression of selectable marker genes and/or foreign genes of interest in rice.
Plastid transformation in rice is highly desirable. The present example provides compositions and methods to effectuate rice plastid transformation in embryogenic cultured rice cells. Such cells may be efficiently regenerated into mature plants (Vasil, I K (1994) Plant Mol Biol 25:925-937; Christou, P (1996) Trends Plant Sci 1:423-431). Data from cultured tobacco (BY2) cells suggest that plastid promoter utilization in tissue culture may be different from those in leaves (Vera A, Sugiura M (1995) Curr Genet 27:280-284; Vera A, Hirose T, Sugiura M (1996) Mol Gen Genet 251:518-525 et al., 1996; Kapoor S, Suzuki J Y, Sugiura M (1997) Plant J 11:327-337). The data presented herein reveal that cultured embryogenic rice cells and leaves utilize the same promoter. rbcL, atpB, 16SrDNA and clpP have only one promoter each which is recognized by the plastid-encoded plastid RNA polymerase (PEP). In contrast, clpP is transcribed by the nucleus-encoded plastid RNA polymerase (NEP) in both samples. The RNAs for clpP and 16SrRNA are relatively abundant in embryogenic cells indicating that the promoters of these genes may be suitable to drive the expression of selectable marker genes.
Materials and methods for Example IV
Plant Materials. Embryogenic rice callus was initiated from mature seed of cv. Taipei 309 on LS2.5 medium (Abdullah R, Cocking E C, Thompson J A (1986) Bio/Technology 4:1088-1090). The calli were introduced into liquid AA medium (Muller A J, Grafe R (1978) Mol Gen Genet 161: 67-76) to establish embryogenic suspension cultures, and subcultured at biweekly intervals. DNA and RNA were prepared from 3 month old cultures 14 days after subculture. Plants were regenerated from embryogenic calli on complete MS medium supplemented with 2 mg/L BAP and 3% sucrose (Murashige T, Skoog F (1962) Physiol Plant 15: 473-497 1962) and transferred onto hormone-free MS medium. Leaves for the isolation of nucleic acids were taken from these plants after four months.
RNA and DNA gel blots. Total cellular RNA was prepared according to Stiekema et al., 1988, supra. The RNA (5 μg per lane) was subjected to electrophoresis in a formaldehyde-agarose gel, blotted and hybridized (Hajdukiewicz P T J, Allison L A, Maliga P (1997) EMBO J 16:4041-4048). Double-stranded ptDNA probes were prepared by random-primed 32P-labeling of PCR-generated or gel-purified DNA fragments. The sequence of the primers used for PCR, along with their positions within the tobacco (N.t.; accession no. Z00044; Shinozaki et al. 1986, supra) or maize (Z.m.; accession no. X86563; Maier et al. 1995, supra) ptDNA are as follows:














5′ nt





position



in

SEQ



plastid

ID


Gene
DNA
Sequence
NO:



















atpB
(Z.m.)
55860 (C)
GAGAGGAATGGAAGTGATTGACA
(33)




55103
GAGCAGGGTCGGTCAAATC
(34)


clpP
(Z.m.)
69840
ATCCTAGCGTGAGGGAATGCTA
(35)




70064 (C)
AGGTCTGATGGTATATCTCAGTAT
(36)









The following ptDNA fragments were used as probes: rbcL (N.t.), a BamHI fragment (nucleotides 58047 to 59285 in ptDNA); 16SrDNA (N.t.), EcoRI to EcoRV fragment (nucleotides 138447 to 140855).
The probe for tobacco 25S rRNA was from plasmid pKDR1 (Dempsey et al., 1993, supra) containing a 3.75 kb EcoRI fragment from a tobacco 25S/18S locus cloned in plasmid pBR325.
Total leaf DNA for relative plastid genome copy number determination was prepared (Mettler I J (1987) Plant Mol Biol Rep 5:346-349), digested with the EcoRI restriction endonuclease, separated on 0.7% agarose gels, blotted and hybridized with the plastid 16SrDNA and cytoplasmic 25SrDNA probes (Allison et al., 1996, supra).
Primer extension analysis. Primer extension reactions were carried out on total leaf RNA as described (Allison and Maliga, 1995, supra). The primers are listed below, with nucleotide position in the published rice plastid genome sequence (Hiratsuka et al., 1989, supra).














5′ nt





position in


Gene
plastid DNA
Sequence
SEQ ID NO:







rbcL
 54124 (C)
ACTTGCTTTAGTTTCTGTTTGTGGTGACAT
(60)


atpB
 53287
AGAAGTAGTAGGATTGGTTCTCATAAT
(61)


16S rRNA
123777
CCGCCAGCGTTCATCCTGAGC
(62)


clpP
 68263
GGTACTTTTGGAACACCAATGGGCAT
(39)









Primer extension reactions were carried out with 1 μg of RNA from leaves, and 10 μg (clpP, 16SrDNA) and 30 μg (rbcL, atpB) of RNA from embryogenic cells.

Results and Discussion

Several plastid promoters have been identified in rice and related monocots. To assess whether these promoters would be suitable to drive expression of selectable marker genes and/or foreign genes of interest in these plant species, transcript accumulation was examined. The rbcL gene in rice and maize is transcribed from a PEP promoter (Mullet et al., 1985, supra; Nishiziwa Y, Hirai A (1987) Jpn J Genet 62:389-395). The atpB gene in maize chloroplasts is transcribed from a PEP promoter (Mullet et al., 1985, supra), whereas in maternal white iojap seedlings lacking PEP it is transcribed from an alternative NEP promoter. The 16SrDNA gene (the first gene of the plastid ribosomal RNA operon) in barley chloroplasts is transcribed from a PEP promoter (Reinbothe S, Reibothe C, Heintzen C, Seidenbecher C, Parthier B (1993) EMBO J 12:1505-1512), whereas in the white albostrians seedlings lacking PEP it is transcribed from an uncharacterized NEP promoter (Hess et al., 1993, supra). The clpP gene in wild-type and iojap maize chloroplasts is transcribed from a NEP promoter.
To determine the level of expression of these genes in embryogenic rice cells and leaf cells, accumulation of mRNAs was assessed on Northern blots. The data reveal that transcript levels in embryogenic cells relative to leaves were barely detectable for rbcL (153-times lower), reduced for atpB (37-fold lower) and 16SrDNA (7-fold lower), and similar for clpP (approximately 1.1-times lower). See FIG. 14. Interestingly, the number of plastid genome copies (ptDNA) per cell is about the same in embryogenic cells and leaves. See FIG. 15. Accordingly, the differences in transcript levels represent values normalized for ptDNA copy number.
To further characterize active plastid promoters, transcript 5′ ends were mapped by primer extension in cultured embryogenic cells and in leaves. Two different 5′ ends, at 312 and 58 nucleotides upstream of the translation initiation codon, were mapped for rbcL. See FIG. 16. The same two 5′-ends were identified in leaf chloroplasts, and in the plastids of embryogenic cells. Two 5′-ends were mapped to similar positions by S1 nuclease analysis in rice chloroplasts (Nishizawa and Hirai, 1987, supra). The rbcL −312-end is downstream of −10/−35 σ70-type promoter elements which are conserved in monocots; the −58-end is generated by RNA processing (Mullet et al., 1985, supra; Reinbothe et al., 1993, supra).
For atpB, a single 5′-end was mapped 310 nucleotides upstream of the translation initiation codon. As for rbcL, the same 5′-ends were identified in embryogenic cells and leaves. See FIG. 16. The −310 end is associated with PEP promoter elements, and has been reported earlier in chloroplasts for rice (Nishizawa Y, Hirai A (1989) Jpn J Genet 64:223-229) and maize (Mullet et al., 1985, supra).
For the rRNA operon, the same two 5′ ends were mapped upstream of the mature 16SrRNA in embryogenic cells and in leaves shown in FIG. 16. Based on DNA sequence conservation, the −116 end is the product of a PEP promoter whereas the −28 end derives from RNA processing. See FIG. 17A; Strittmatter G, Godzicka-Josefiak A, Kossel H (1985) EMBO J 4:599-604; Vera and Sugiura, 1995, supra; Allison et al., 1996, supra).
For clpP, the same 5′ end was mapped in embryogenic tissue culture cells and in leaves. See FIG. 16. The transcript initiates 111 nucleotides upstream of the translation initiation codon within the 10-nucleotide NEP consensus, shown in FIG. 5A, and is the product of the clpP Type-I NEP promoter.
Mapping of RNA 5′ ends upstream of rbcL, atpB, 16SrDNA and clpP identified the same promoters in cultured embryogenic cells and in the leaves of rice. The data obtained from these studies in rice can be contrasted with the results reported for tobacco. In tobacco, atpB and 16SrDNA are preferentially transcribed from PEP promoters in leaves, and from NEP promoters in BY2 tissue culture cells (Vera and Sugiura 1995; Kapoor et al. 1997). In rice embryogenic cultures, no PEP to NEP promoter switch was observed for any of the plastid genes examined. The data suggest that a PEP to NEP promoter switch is not essential for adaptation to cell culture. Alternatively, atpB and 16SrDNA, genes which have PEP and NEP promoters in other monocots as discussed in the previous examples, have no NEP promoters in rice.
One important difference between embryogenic rice cells and the tobacco BY2 cell line is the length of time in culture. The rice cell culture line described herein is only a few month old and has maintained the ability to regenerate plants. The BY2 cell line has been grown in culture for several years and has lost the capacity for plant regeneration (Yasuda T, Kuroiwa T, Nagata T (1988) Planta 174:235-241). Accordingly the possibility remains that in BY2 cells plastid gene expression may be different from tobacco cells in a short-term culture or in a tobacco plant.
The results presented herein provide practical implications for the production of transgenic rice cells and or plants. Two powerful promoters are described herein which are active in rice embryogenic cells: the clpP NEP promoter and the rrn PEP promoter. Both promoters are suitable to drive the expression of selectable marker genes for plastid transformation in embryogenic rice cells.
While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

## Claims

1. A DNA construct for stably transforming the plastids of higher plants, comprising:
a) a transcription unit encoding at least one exogenous protein of interest; and
b) a NEP promoter and a PEP promoter in tandem, operably linked to said transcription unit, wherein expression of said transcription unit is regulated by said promoters,
wherein said NEP promoter is the NEP promoter from a gene selected from the group consisting of clpP, rpoB, and atpB, wherein said atpB gene is from a plant selected from the group consisting of maize, sorghum, barley, wheat, and rice, wherein said rpoB gene is selected from the group consisting of maize, and rice, and wherein said clpP gene is from a plant selected from the group consisting of maize, rice, and wheat; and
wherein said PEP promoter is Prrn or is the clpP PEP promoter from rice.

2. A DNA construct for stably transforming the plastids of higher plants, comprising:
a) a transcription unit encoding at least one exogenous protein of interest; and
b) a NEP promoter and a PEP promoter in tandem, operably linked to said transcription unit, wherein expression of said transcription unit is regulated by said promoters, wherein said NEP promoter is clpP −111 and said PEP promoter is Prrn.

3. The DNA construct according to claim 2, wherein said Prrn has the sequence of SEQ ID NO: 32.

4. A DNA construct for stably transforming the plastids of higher plants, comprising:
a) a transcription unit encoding at least one exogenous protein of interest; and
b) a NEP promoter and a PEP promoter in tandem, operably linked to said transcription unit, wherein expression of said transcription unit is regulated by said promoters, wherein said NEP promoter is clpP −53 and said PEP promoter is Prrn.

5. The DNA construct according to claim 4, wherein said Prrn has the sequence of SEQ ID NO: 32.