Plant Mol Biol (2009) 70:487–498 DOI 10.1007/s11103-009-9486-x
Next generation synthetic vectors for transformation of the plastid genome of higher plants Sugey Ramona Sinagawa-Garcı´a Æ Tarinee Tungsuchat-Huang Æ Octavio Paredes-Lo´pez Æ Pal Maliga
Received: 13 January 2009 / Accepted: 29 March 2009 / Published online: 23 April 2009 Ó Springer Science+Business Media B.V. 2009
Abstract Plastid transformation vectors are E. coli plasmids carrying a plastid marker gene for selection, adjacent cloning sites and flanking plastid DNA to target insertions in the plastid genome by homologous recombination. We report here on a family of next generation plastid vectors carrying synthetic DNA vector arms targeting insertions in the rbcL-accD intergenic region of the tobacco (Nicotiana tabacum) plastid genome. The pSS22 plasmid carries only synthetic vector arms from which the undesirable restriction sites have been removed by point mutations. The pSS24 vector carries a c-Myc tagged spectinomycin resistance (aadA) marker gene whereas in vector pSS30 aadA is flanked with loxP sequences for post-transformation marker excision. The synthetic vectors will enable direct manipulation of passenger genes in the transformation vector targeting insertions in the rbcL-accD intergenic region that contains many commonly used restriction sites. Keywords Homologous recombination Nicotiana tabacum Plastid transformation Tobacco Vector
Electronic supplementary material The online version of this article (doi:10.1007/s11103-009-9486-x) contains supplementary material, which is available to authorized users. S. R. Sinagawa-Garcı´a T. Tungsuchat-Huang P. Maliga (&) Waksman Institute, Rutgers, The State University of New Jersey, 190 Frelinghuysen, Road, Piscataway, NJ 08854-8020, USA e-mail:
[email protected] S. R. Sinagawa-Garcı´a O. Paredes-Lo´pez Depto. de Biotecnologı´a y Bioquı´mica, Centro de Investigacio´n y de Estudios Avanzados del IPN, Apdo. Postal 629, 36500 Irapuato, Gto., Mexico
Introduction The *150-kb plastid genome (ptDNA) of higher plants is highly polyploid (Palmer 1985; Wakasugi et al. 2001). Tobacco (Nicotiana tabacum) cells, dependent on the cell type, contain 1,000–10,000 ptDNA copies compartmentalized in 10–100 plastids (Shaver et al. 2006; Thomas and Rose 1983). Because of readily obtainable high protein levels, the opportunity to express operons and natural containment, the plastid genome is an attractive target for biotechnological applications (Bock 2007; Daniell et al. 2005; Maliga 2003). Plastid transformation in higher plants was reported in 1990 in tobacco (Svab et al. 1990). Other than tobacco, plastid transformation is routine in only a handful of crops for example in tomato (Ruf et al. 2001), soybean (Dufourmantel et al. 2004) and lettuce (Kanamoto et al. 2006; Lelivelt et al. 2005). Transformation of the plastid genome in higher plants relies on homologous recombination. Plastid transformation vectors are high copy E. coli plasmids that carry ptDNA sequences flanking a selectable marker gene, most commonly encoding spectinomycin (aadA) or kanamycin resistance (neo or aphA-6) and a useful gene that lacks a selectable phenotype. The marker gene and the passenger gene are referred to here as the vector load. Plastid vectors lack sequences that would sustain long-term replication in plastids. Thus, when the transforming DNA is introduced into plastids by the biolistic process or by polyethylene glycol treatment, marker-encoded antibiotic resistance may be expressed only if the marker gene is incorporated in the plastid genome. Cells carrying the transformed plastid genome are cultured on a selective medium to allow sufficient time to dilute out the non-transformed ptDNA copies then regenerated into plants. The regenerated plants are often chimeric because, once a shoot apex with slowly
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dividing long-term stem cells is formed, sorting out of transgenic and non-transgenic ptDNA is slowed down (Lutz and Maliga 2008). One or two additional cycles of shoot (plant) regeneration from somatic cells are routinely employed to ensure that all somatic and germline cells are homoplastomic. For general reviews on plastid transformation see references (Bock 2001; Koop et al. 2007; Maliga 2004; Verma and Daniell 2007). Advanced plastid vectors carry sequences for posttransformation removal of the marker gene and a polycloning site for incorporation of the gene of interest in the vector. A two-step process for efficient excision of plastid markers utilizes phage site-specific recombinases (Lutz and Maliga 2007). First transplastomic plants are obtained with recombinase recognition sequences flanking the plastid marker genes, such as the P1 phage loxP site or the phiC31 phage attB/attP sites. The transplastomes carrying the recombinase recognition sites are stable in the absence of Cre or Int recombinases. However, when a suitably engineered Int or Cre is introduced, the plastid marker is simultaneously excised from all ptDNA copies. The second feature of advanced plastid vectors, a polycloning site, facilitates assembly of passenger genes in the plastid vector. If the plastid targeting sequences in the vector lack commonly used restriction sites, the plastid vector can replace the E. coli cloning vectors so that a gene assembled in the plastid vector can be directly used for plastid transformation. Examples for such vectors are the pPRV vectors targeting the inverted repeat region (Lutz et al. 2007; Zoubenko et al. 1994) and the pRB94 and pRB95 vectors (Ruf et al. 2001) and their pZF derivatives (Zhou et al. 2007) targeting the trnfM and trnG intergenic region in the large single copy region of the plastid genome. Fig. 1 Predicted secondary structure of mRNA downstream of rbcL generated by the GCG FoldRNA program (Zuker et al. 1991) and displayed as a squiggles output file. We marked the position of the rbcL TAA stop codon (included in the sequence), the position of processed rbcL 30 -end and the StuI and ScaI insertion sites. The accD start codon is 876 nucleotides downstream of the rbcL stop codon and is not shown
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We report here that genomic regions with many restriction sites may be conveniently targeted for insertion if synthetic DNA is used as targeting sequence from which the undesirable restriction sites have been removed by point mutations. The synthetic vector we tested targets the rbcL-accD intergenic region in the large unique region of the tobacco ptDNA. This site was the first region transformed with a chimeric aadA gene (vector pZS197; Svab and Maliga 1993). Although derivatives of the pZS197 targeting region are still used in plastid transformation vectors (Birch-Machin et al. 2004; Monde et al. 2000a; Wostrikoff and Stern 2007; Zhou et al. 2008) the many restriction sites in the targeting region make vector construction tedious. A second problem of vector pZS197 is the opportunistic insertion site between two adjacent BamHI sites that, when transcribed into RNA, form a potential stem-loop structure (loop marked with StuI site in Fig. 1). Transgenes inserted downstream of rbcL in a direct orientation apparently interfere with rbcL mRNA maturation, yielding complex polycistronic mRNAs with rbcL as the first reading frame in some (Monde et al. 2000a; Staub and Maliga 1994) or all transcripts (Staub and Maliga 1995a). The insertion site in the new synthetic vector was moved downstream of the repeat (ScaI site in Fig. 1) to facilitate processing of the rbcL mRNA. We report that recombination between genomic DNA and vector targeting arms most often takes place at the ends of homologous regions yielding plants with entirely synthetic or entirely wild type ptDNA in the targeted region. The pSS22 vector backbone is an open construct that may be used to develop custom expression vectors targeting insertions in the rbcL-accD intergenic region. The pSS24 synthetic vector carries a c-Myc tagged spectinomycin
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resistance (aadA) marker gene whereas in vector pSS30 aadA is flanked with loxP sequences for post-transformation marker excision. In the transplastomic plants wild-type rbcL mRNA maturation may be ensured using appropriate designs.
Materials and methods Construction of transformation vectors Plasmid pSS16 is a pENTR221 (Invitrogen Life Technologies, Gateway Entry Vector, Carlsbad, CA) derivative, carrying the synthetic rbcL-accD plastid-targeting region as a 3.1-kb SwaI fragment purchased from Blueheron Biotechnology (Bothell, WA). Plasmid pSS22 is a pUC119 vector derivative lacking the ScaI site, in which the PvuII fragment was replaced with the SwaI fragment from plasmid pSS16 (GenBank Accession No. FJ416604). Plasmid pSS24 is a plasmid pSS22 derivative carrying a c-Myc tagged spectinomycin resistance (aadA) gene in a PpsbATpsbA cassette (Lutz et al. 2007) and lacking the NruI-NsiI fragment (GenBank Accession No. FJ416605). Plasmid pSS30 is a plasmid pSS22 derivative in which the aadA gene is flanked by the P1-phage loxP sites (GenBank Accession No. FJ416606). Plastid transformation vector pSS25 is a plasmid pSS24 derivative digested with PmlI and HindIII and re-ligated to remove the PmlI and HindIII restriction sites and the recognition sequences between the two sites. Plasmid pSS31 is a plasmid pSS30 derivative, in which the PmlI and HindIII restriction sites and the recognition sequences between the two sites have been removed. Plastid vector pSS45 is a pSS24 plasmid derivative carrying an PrrnLatpB::neo (SacI-XbaI fragment) insert cloned into a SacI-XbaI digested vector. The PrrnLatpB::neo fragment derives from plasmid pHK30 (Kuroda and Maliga 2001) in which the NheI site has been removed (Lutz et al. 2007). Plastid vector pSS46 is a pSS30 derivative carrying the PrrnLatpB::neo (SacI-XbaI) fragment present in pSS45. Plastid transformation Transformation of the tobacco (Nicotiana tabacum cv. Petit Havana) plastid genome was carried out as previously described (Lutz et al. 2006; Svab and Maliga 1993). DNA for plastid transformation was prepared using the QIAGEN Plasmid Maxi Kit (QIAGEN Inc., Valencia, CA). Transforming DNA was introduced into leaf chloroplasts on the surface of 0.6 lm gold particles using the Du Pont PDS1000He biolistic gun. Transplastomic plants were selected on RMOP medium containing 500 mg l-1 spectinomycin dihydrochloride. The transgenic plants were
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grown on MS (Murashige-Skoog) medium (Murashige and Skoog 1962) containing 3% (w/v) sucrose and 0.7% (w/v) agar in sterile culture condition. A uniform population of transformed plastid genome copies was confirmed by DNA gel blot analysis using the rbcL-accD plastid targeting region probe, the 3.1-kb SwaI synthetic ptDNA fragment excised from plasmid pSS16. Double-stranded DNA probes were prepared by random-primed 32P-labeling using the Amersham Ready-To-Go DNA Labeling Beads (Amersham Biosciences Corp., Piscataway, NJ). RNA gel blot analysis RNA gel blot analysis was carried out as described (Silhavy and Maliga 1998). Briefly, total cellular RNA was prepared from leaves (Stiekema et al. 1988) and the RNA (5 mg per lane) was electrophoresed on 1% agarose/ formaldehyde gels, and then transferred to Hybond N membranes (Amersham) using the posiblot transfer apparatus (Stratagene). Hybridization to random-primer labeled fragment was carried out in a modified Church hybridization buffer (0.5 M phosphate buffer, pH 7.2, 10 mM EDTA, 7% SDS (Church and Gilbert 1984) overnight at 65°C. Radioactive probes were prepared by randomprimed 32P-labeling (see above) of gel-purified DNA fragments of the following genes: rbcL, 1.4-kb SwaI-BglII fragment from plasmid pSS16 (see above); aadA, 0.8-kb NcoI-XbaI fragment from plasmid pHC1 (Carrer et al. 1990); accD, 0.7-kb BstBI-SwaI fragment from plasmid pSS16 (GenBank Accession No. FJ416604); and neo, 0.7-kb NheI-XbaI fragment from plasmid pHK30 (Kuroda and Maliga 2001). Testing the rbcL-accD region in transgenic ptDNA Total cellular DNA was isolated by the CTAB protocol and the left and right targeting regions were amplified by PCR and digested with the appropriate restriction enzymes. The left targeting region was amplified with the following primers: in plants transformed with plasmids pSS25 and pSS31, 50 -CACGGAATTCGTGTCGAGTAGACC-30 and 50 -CGCTCGATGACGCCAACTACC-30 ; in plants transformed with plasmids pSS45 and pSS46, 50 -CACGGAATT CGTGTCGAGTAGACC-30 and 50 -TGACAGCCGGAAC ACGGCGGC-30 . The right targeting region in all transgenic plants was amplified with primers 50 -CCTGCCGG CCCAGTATCAGCCC-30 and 50 -CCATAGGATCCCAA GTACCCGG-30 . The thermal cycling conditions were 2 min denaturation at 94°C followed by 25 cycles of amplification (45 sec at 94°C, 45 sec at 60°C, 1 min at 72°C). Diagnostic restriction sites tested in targeting region are listed in Supplementary Table S1.
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SDS-PAGE and immunoblotting SDS-PAGE and immunoblotting were carried out essentially as described (Chakrabarti et al. 2006). Briefly, leaves for protein extraction were taken from greenhouse plants. To obtain total soluble leaf protein, about 200 mg leaf was homogenized in 0.1 ml buffer containing 50 mM HEPES/ KOH (pH 7.5), 10 mM potassium acetate, 5 mM magnesium acetate, 1 mM EDTA, 10 mM DTT, 2 mM PMSF and 5 mg/ml Na-L-ascorbate. Protein concentrations were determined by the Bradford protein assay reagent kit (BioRad, Hercules, CA), separated in SDS-PAGE and stained in Comassie brilliant blue R-250 solution. Immunoblot analysis of aminoglycoside 3’’-adenylyltransferase (AAD), was carried out as described (Carrer et al. 1993) using Fig. 2 Mapping recombination sites in the transplastomes. a Left and right targeting regions (LTR and RTR) of the synthetic vectors with ptDNA polymorphic sites. Only restriction sites used for mapping synthetic vector sequences in the transplastomes are shown. Below the map are listed the number of mismatched nucleotides and insertion/deletion loops (IDLs) the length of which is given in nucleotides. The synthetic rbcLaacD plastid-targeting region shown here is found in vectors pSS24 (GenBank Accession No. FJ416605) and pSS30 (GenBank Accession No. FJ416606). b Synthetic vector sequences in the transplastomic lines. Regions derived from wild-type ptDNA (top) and the synthetic vector (bottom) is indicated as heavy lines. The recombination sites are marked with dashed lines and the triangles symbolize loxP sites. The origin of *1 kb RTR could not be determined in the absence of markers and is marked with a heavy line in both vector and wild-type ptDNA maps. Primary data on digestion of PCR fragments are listed in supplementary Table S1
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commercial c-Myc antibody (1:1,100) purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Results Synthetic vectors targeting insertions in the rbcL-accD intergenic region The transformation vectors carry a 3-kb synthetic DNA fragment as plastid targeting sequence (Fig. 2a) including the 1.4-kb rbcL coding region (with the exception of the first two codons), the rbcL-accD intergenic region (764 nucleotides) and 0.8 kb of the 1.5 kb accD coding region. Silent point mutations in the tobacco ptDNA
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(GenBank accession no. Z00044) were introduced to remove 16 commonly used restriction sites in the rbcL (13 sites) and accD (three sites) coding regions. In the intergenic region four restriction sites (AccI, XbaI and 2xBamHI) have been removed by point mutations. In addition, we designed three potential insertion sites: The BglII site downstream of rbcL, a StuI site at the location used in vector pZS197 and the new ScaI insertion site at a location where we expected no interference with rbcL mRNA processing (Fig. 1, 2a). In the synthetic vectors the ScaI site divides the ptDNA into the 1.8-kb Left and 1.2-kb right targeting regions (LTR and RTR, respectively). Restriction site polymorphisms shown in Fig. 2a enabled identification of vector sequences incorporated into the plastid genome with the vector load. The synthetic plastid-targeting region was purchased with restriction sites to accommodate insertions at any of the three potential target sites. Plasmid pSS22 is an intermediate vector shown in Fig. 3. Plasmids pSS24 and pSS30 (Fig. 3) are pSS22 derivatives lacking most restriction sites downstream of the rbcL gene and carry a selectable spectinomycin resistance (aadA) marker gene. In vector pSS30 loxP sites flank aadA to facilitate posttransformation excision of the marker gene. Plastid transformation vectors pSS24 and pSS30 have a cluster of unique cloning sites which, when transcribed into mRNA, may fold into secondary structures. We therefore digested plasmids pSS24 and pSS30 with the PmlI and HindIII restriction enzymes and ligated the ends to obtain
Fig. 3 Plastid vectors pSS22 (GenBank Accession No. FJ416604), pSS24 (FJ416605) and pSS30 (FJ416606) with synthetic plastidtargeting regions. Shown are the rbcL and accD plastid targeting sequences, cloning sites, the aadA marker gene with the C-terminal cmyc tag (black box) and loxP sites (black triangles). Promoter (P) and Terminator (T) sequences are marked for rbcL (T1; native element), aadA (P2, T2; derived from plastid psbA gene) and accD (P3; native element)
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plasmids pSS25 and pSS31, respectively (Fig. 4). Derivatives with a kanamycin resistance passenger gene were obtained by cloning a promoter-neo coding region fragment upstream of the aadA marker gene to obtain vectors pSS45 and pSS46 (Fig. 5). The four plasmids were introduced into chloroplasts by the biolistic process where the vector load integrated into the plastid genome via the homologous targeting sequences. Transplastomic clones were selected by spectinomycin resistance and uniform transformation of ptDNA was verified by DNA gel blot analyses (data not shown). Ten bombardments with vectors pSS25, pSS31, pSS45 and pSS46 yielded 11, 15, 23 and 19 transplastomic clones, respectively. Transplastomic clones derived from an independent transformation event were identified by the plasmid name and a serial number, for example Nt-pSS25-1C. The letter at the end identified an independently regenerated shoot from the same initial event termed a subclone. Plants Nt-pSS25-10B and Nt-pSS25-10C derive from the same initial event, but represent two independently regenerated shoots. Two letters after the serial number refer to two cycles of plant regeneration to obtain homoplastomic plants. Incorporation of vector sequences in the plastid genome The marker gene and the gene of interest integrate into the plastid genome by two homologous recombination events via the targeting sequences. If recombination occurred at the end of the targeting region, transformation resulted in replacement of the endogenous ptDNA with synthetic vector sequences. However, if recombination occurred adjacent to the marker gene, no synthetic vector sequence is present in the transplastome. To identify the origin of ptDNA in our transplastomic plants, we PCR-amplified the targeting region from the transplastomes and subjected the PCR fragments to digestion with diagnostic restriction enzymes. We analyzed plants representing 12 independently transformed clones and a total of 20 independently regenerated plants (Fig. 2b, Supplementary Table S1). We report here that vector LTR sequences incorporated in the plastid genome in *50% (6 out of 12) of the clones. In five clones the entire targeting region was replaced with synthetic sequences whereas in one clone, pSS31-15, partial replacement occurred. Multiple plants (subclones) regenerated from the same clone had sequences of the same origin in the targeted region. The only exceptions were subclones regenerated from transformation event Nt-pSS45-8. Subclone Nt-pSS45-8B carries the vector LTR whereas the second plant, Nt-pSS45-8A carries the cognate wild-type ptDNA. Recovery of multiple transplastomes in the same lines could be the products of multiple independent transformation events (Carrer and Maliga 1995) or secondary gene conversion between
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transgenic and wild type ptDNA (Khakhlova and Bock 2006; Staub and Maliga 1995b). The ScaI sites are present in each of the clones flanking the marker gene because they are part of the heterologous block that is absent in the wild-type ptDNA. There are only three markers to probe for the origin of the 1.2-kb RTR close to the right end: the AccI, XhoI and SspI sites are 163 nt, 106 nt and 17 nt from the right end, respectively. Because there are no markers in the *1 kb region between the ScaI site (adjacent to marker gene) and the AccI site (163 nt from the RTR right end), we can conclude only that recombination in all clones but one occurred in this *1 kb region. This shared region is marked with a heavy line in Fig. 2b in both vector and wild-type ptDNA maps. In clone Nt-pSS25-1C the RTR region derives from the vector except the SspI marker that is 17 nt from the RTR right end. The absence of SspI site deletion caused by a T to C change 17 nt from the right end is likely to be fortuitous. Fig. 4 Expression of the aadA marker gene in the synthetic vector. a Monocistronic mRNA accumulates from the plastid rbcL and accD genes in wild type tobacco plastids. Shown is the partial map of the ptDNA with the rbcL and accD genes. The transcripts are depicted as wavy lines above the genes. The supporting RNA gel blots are shown below the map. b Monocistronic mRNA accumulates from aadA in plastids transformed with plasmid pSS25 (Clone NtpSS25-10C). Processing of rbcL and accD mRNAs is not affected in the transplastomic plants c The loxP-sites flanking aadA interfere with mRNA maturation in plastids transformed with plasmid pSS31 (Clone Nt-pSS31-15C). Note accumulation of dicistronic rbcL-aadA and aadA-accD mRNAs. 5 lg RNA prepared from the youngest to the oldest leaf of greenhousegrown plants was loaded in lanes 1–5. Promoter (P) and Terminator (T) sequences are marked for rbcL (P1, T1; native elements), aadA (P2, T2; derived from plastid psbA gene) and accD (P3, T3; native elements)
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The aadA marker gene in the plastid genome does not interfere with RNA maturation The rbcL and accD mRNAs in wild type tobacco plastids are monocistronic (Fig. 4a). RNA gel blot analysis was carried out to determine whether or not moving the insertion site farther downstream eliminates interference with processing of the rbcL mRNA in transplastomic plants. To this end total leaf RNA was prepared from leaves of NtpSS25-10C plants, separated in agarose-formamide gels and hybridized with the rbcL, aadA and accD probes (Fig. 4b). The leaves were harvested from plants not yet flowering and 10–15 cm in height. Because mRNA abundance and processing may be affected by leaf age, we prepared RNA from four to five consecutive leaves from young through fully expanded to mature in age. Blots in Fig. 4b indicate that only monocistronic mRNAs accumulate for rbcL, aadA and accD. Thus, moving the
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insertion sites from 271 (as in vector pZS197) to *410 nucleotides downstream of rbcL was necessary and sufficient for efficient processing of mRNA at 141 nucleotides downstream of the stop codon. Interestingly, the aadA mRNA in Nt-pSS25-10C leaves is monocistronic. The aadA gene in this construct has TpsbA, an *200 bp sequence derived from downstream of the plastid psbA gene (Fig. 4b) that is sufficient for efficient processing of the aadA mRNA. Processing of psbA mRNA normally occurs 93 nucleotides downstream of the stop codon (Shinozaki et al. 1986).
Complex processing of loxP-containing mRNAs We were interested to characterize mRNA accumulation in plants transformed with the synthetic vector in two common applications: post-transformation excision of the marker gene and integration of a useful gene in the plastid genome. Cis elements incorporated in vector pSS31 for marker excision are the loxP sites flanking the aadA marker gene. RNA isolated from pSS31-transformed plants was therefore subjected to gel blot analyses to determine whether or not incorporation of loxP sequences affect mRNA processing. Surprisingly, we found rbcL-aadA and aadA-accD
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dicistronic mRNAs (Fig. 4c) indicating that the stem-loop structure encoded in the loxP sites affects both, rbcL and aadA mRNA processing. The second application, incorporation of a useful gene in the plastid genome, was modeled by inserting a kanamycin resistance (neo) gene upstream of aadA. The neo gene was introduced in the plastid genome in two vectors, one carrying aadA with and without the loxP sites. In plants transformed with the neo-aadA construct (Nt-pSS451AA) monocistronic rbcL and accD mRNAs accumulated (Fig. 5a). Thus, inserting an additional (passenger) gene upstream of aadA did not significantly affect mRNA maturation. The neo gene in Fig. 5a has its own promoter but no sequences downstream of the coding region to facilitate processing of the neo mRNA. As expected, we detected only a 2-kb dicistronic neo-aadA mRNA with the neo probe and a monocistronic aadA (1 kb) and dicistronic neo-aadA transcript with the aadA probe (Fig. 5a). Interestingly, the highest is the neo mRNA level in older pSS45 leaves, whereas it is the lowest in the older pSS46 leaves apparently due to the presence of loxP sites (Fig. 5). Including the neo passenger gene upstream of the floxed aadA had a dramatic effect, facilitating normal maturation of both rbcL and accD mRNAs (Nt-pSS46-14BC plants, Fig. 5b). The neo probe detected only the 2-kb dicistronic neo-aadA mRNA, whereas the aadA probe detected both
Fig. 5 Transcript accumulation in chloroplasts transformed with vector carrying a gene of interest. a Expression of dicistronic mRNA in plants transformed with vector pSS45 (clone Nt-pSS45-1AA) does not significantly affect processing of mRNA transcribed from the flanking rbcL and accD genes. b No significant amounts of readthrough transcripts including rbcL accumulate from floxed aadA in vector pSS46 (clone Nt-pSS46-14BC) when neo is inserted upstream of aadA. 5 lg RNA prepared from the youngest to the oldest leaf of greenhouse-grown plants was loaded in lanes 1–4. Promoter (P) and Terminator (T) sequences are marked for rbcL (P1, T1; native elements), neo (P4, PrrnLatpB promoter), aadA (P2, T2; derived from plastid psbA gene) and accD (P3, T3; native elements)
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the monocistronic aadA (1 kb) and the dicistronic neoaadA transcript, as expected. NPTII and AAD accumulation in the transplastomic plants We have chosen the PrrnLatpB::neo as a passenger gene because the expression of its progenitor has been well characterized in Nt-pHK30 transplastomic plants. The NtpHK30 plants accumulated NPTII in leaves at about 7% of the total soluble cellular protein (TSP; Kuroda and Maliga 2001). The construct used in the present study was modified by: removing an NheI site 13 amino acids downstream of the start codon; the neo gene used here has no 30 UTR
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downstream of the coding region; and the gene was inserted in the large single copy region rather than in the inverted repeat of the plastid genome. However, the pHK30 50 translation control region is preserved and the dicistronic mRNA has a stabilizing 30 UTR, which are the elements that are essential for protein accumulation (Maliga 2003). Thus, we were not surprised that NPTII accumulation could be readily detected in Nt-pSS45 and Nt-pSS46 plants in Coomassie brilliant blue R250 stained SDS-PAGE gels (Fig. 6a). Detecting a protein in stained gels requires protein accumulation in excess of 1% of the TSP. AAD, the protein product of the aadA marker gene is below detection level in the transplastomic plants in Coomassie stained gels. However, AAD could be readily detected by the c-Myc antibody (Fig. 6b). The AAD levels appear to correlate with mRNA levels.
Discussion Transgene insertion in the rbcL-accD intergenic region by homologous recombination via synthetic vector arms
Fig. 6 Protein accumulation from the neo and aadA transgenes in chloroplasts. a NPTII (30.4 kDa) detected by Coomassie brilliant blue R250 in SDS-PAGE gel. 50 lg total soluble protein was loaded per lane. Marked are the ribulose-1,5-bisphosphate carboxylase/oxygenase large (LSU) and small (SSU) subunits. b Immunoblot analysis to detect AAD (32 kDa) by the c-myc tag. 100 lg total soluble protein was loaded per lane
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We report here targeted insertion of transgenes into the rbcL-accD intergenic region with vectors carrying synthetic targeting regions. Because the synthetic vector lacks most major restriction sites from its backbone, it is suitable for direct assembly of passenger genes in E. coli. Since information on integration of sequences differing in single nucleotide mismatches is scanty and they are useful models for mutagenesis by transformation, we studied integration of vector sequences in the transplastomes. Best characterized are the recombination sites in the 1.8-kb LTR where sixteen restriction sites were tested to determine the origin of targeting region in the transplastome (Fig. 2b). It appears that the general rule is recombination close to the ends of plastid targeting regions. When large synthetic LTR sequences were incorporated from the vector, recombination in five out of six clones took place in the 157 nucleotide region between the vector LTR left end and the first (SacII) restriction site in the wild-type ptDNA. In the sixth clone recombination occurred between the SacII and adjacent BglII sites that are 245 nucleotides apart. When ptDNA sequences are found adjacent to the vector load, in five out of six clones recombination took place in the 129nucleotide region between the penultimate StuI site at the right end and the ScaI site marking the beginning of the vector load. In one clone recombination took place further away from the LTR right end, between the wild type AccI site at the 30 -end of rbcL coding region (no BglII site from vector) and the XbaI site downstream of rbcL.
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Recombination close to the ends of the regions of homology suggest heteroduplex formation within the entire region of homology followed by heteroduplex repair using either the wild-type ptDNA or synthetic vector arms as template. As to integration of RTR sequences, in the absence of markers we learned only that most (11 out of 12) recombination events took place within a 1-kb region adjacent to the marker gene. These findings are compatible with incorporation of the vector load by a two-step process. The first step is cointegrate formation by homologous recombination via one of the targeting regions (including heteroduplex repair), followed by a second homologous recombination event via the second targeting region so that the E. coli vector backbone is lost, as proposed for marker excision by the transient cointegration protocol (Klaus et al. 2004). This report extends earlier work on transformation of the plastid genome of tobacco with 6.2-kb homologous ptDNA carrying seven polymorphic markers in vector pJS75. The pJS75 vector did not carry transgenes, only point mutations in plastid genes that conferred spectinomycin and streptomycin resistance. The study of two pJS75-transformed clones indicated that long regions rather than small fragments integrated from the transforming DNA (Staub and Maliga 1992). This study was biased by the necessity to incorporate the point mutations to obtain antibiotic resistance. Because expression of antibiotic resistance is independent of the incorporation of point mutations in this study and the 1.8-kb LTR in the synthetic vectors carries many more DNA polymorphic sites we can now conclude that homologous DNA with a few single nucleotide mismatches and short (?3) insertion/deletion loops will be incorporated in *50% of the clones. Furthermore, the probability of incorporating the point mutations will not be dependent on the distance from the marker gene because recombination is likely to occur at the ends of the homologous regions. The experimentally determined number of transplastomic clones carrying point mutations was lower than the expected value,*25% (3 out of 12 and 5 out of 20, respectively), when mutations were introduced by transformation with aadA vectors carrying engineered targeting regions of the psbE (Bock et al. 1994) and psaB gene (Bock and Maliga 1995). Recombination at the end of the targeting region also facilitated incorporation of heterologous rbcL coding regions in the tobacco plastid genome in 30– 50% of the clones (Kanevski et al. 1999). The pattern of vector targeting sequence integration observed in this study and the references above is significantly different from the integration of partially homologous (homeologous) DNA that integrated into the plastid genome by multiple recombination events via local regions of homology (Kavanagh et al. 1999).
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RNA processing in transplastomic chloroplasts The rbcL mRNA, when transcribed in its native context, is always monocistronic. Because plants transformed with vector pZS197 derivatives contained complex polycistronic rbcL messages (Staub and Maliga 1994) we assumed that transgenes inserted with vector pZS197 interfere with rbcL mRNA 30 -end processing. Stable plastid mRNAs derive from longer transcripts by post-transcriptional cleavage downstream of a stem structure (Monde et al. 2000b). We were thus looking for potential stem-loop structures that could be involved in rbcL mRNA maturation and moved the insertion point downstream of a repeat sequence that could be relevant (Fig. 1). Indeed, aadA at the new insertion site did not interfere with rbcL or accD mRNA maturation and the transplastomic chloroplasts contained monosistronic rbcL and accD mRNAs (Fig. 4b). Flanking aadA with loxP sequences lead to partial processing of rbcL and accD mRNAs (Fig. 4c). However, essentially complete processing of the rbcL and accD mRNAs could be restored when distance between the rbcL 30 processing site and loxP site was increased by inserting neo in the polycloning site (Fig. 5b). In the Nt-pHK105 plastid the stem-loop structure encoded in the loxP site functions as an inefficient mRNA processing site (Tungsuchat et al. 2006). Conflicting effect of loxP sites on mRNA processing is likely to be due to interaction with protein factors associated with flanking sequences. We tested mRNA processing in plants with both synthetic and native LTR and RTR sequences and found no difference in transcript patterns (Figs. 4, 5; unpublished). Also, we did not find any readily detectable phenotypic difference between wild type and transplastomic plants. Protein expression in the synthetic vectors Predictable processing of transgene mRNAs is a desirable objective because the translatability of plastid mRNAs is dependent on mRNA sequences upstream of the AUG translation initiation codon (Maliga 2003). Therefore, the best way to judge the performance of a transgene is to test protein accumulation from a single mRNA species encoding the protein of interest as the only reading frame, or from a mixed population of mRNAs in which the protein of interest is encoded in the first reading frame. Moving the insertion point further downstream enabled essentially complete processing of rbcL mRNA, yielding mRNAs in which the recombinant protein is the first open reading frame. An alternative solution to obtain monocistronic mRNAs in the rbcL-accD intergenic region is insertion of the transgenes convergent to rbcL (Birch-Machin et al. 2004; Zhou et al. 2008). Incorporation of an RNA cleavage
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site between rbcL and the transgene, such as the intercistronic expression element (Zhou et al. 2007) may also be a practical way to obtain stable translatable monocistronic mRNA. Because the new synthetic vectors target insertions in the large single copy region, they are expected to yield less protein than the same gene inserted in the repeated region where the transgenes are present in two copies per plastid genome. Protein expression was checked on Coomassie brilliant blue R250 stained SDS-PAGE gels. NPTII, the passenger gene product, could be readily detected (Fig. 6a). However, we were surprised that AAD, the marker gene product, was not visible on the gel because the PpsbA-TpsbA promoter-terminator expression cassette in which the aadA gene is expressed is expected to yield a few percent protein (Staub and Maliga 1993). Sequencing of PpsbA revealed two point mutations (in lower case below) relative to the tobacco cv. Bright yellow 4 ptDNA sequence in GenBank accession no. Z00044. The cv. Petit Havana sequence in the pSS vectors, starting with the promoter-10 box (underlined) is 50 -TATACTGTTGAATAAcAAt-30 . The A to C change downstream of the-10 promoter element is a polymorphic site in the wild-type cv. Petit Havana ptDNA. The G to T change is present in PpsbA of the pSS vectors only, and is the likely reason for the relatively low level (\1%) AAD accumulation in the transplastomic plants. The G to T mutation occurred in E.coli and remained undetected because the level of expression is sufficient to confer spectinomycin resistance to E. coli and for the recovery of transplastomic clones. Applications of synthetic plastid transformation vectors We did not find obvious phenotypic differences among wild-type plants and transplastomic plants with partial or complete rbcL and accD mRNA processing. However, study of plastid gene knockout lines suggests that impaired plastid gene function may become obvious only under extreme environmental conditions. For example, deletion of the ndhB gene resulted in reduced CO2 fixation under humidity stress (Horvath et al. 2000) and deletion of Rpl33, a non-essential plastid-encoded ribosomal protein, was required only under cold stress conditions (Rogalski et al. 2008). Now that transformation vectors are available that ensure wild-type rbcL and accD transcript maturation, experiments can be designed to test the consequence of partial mRNA processing reported in plastids transformed with pJS and pNT1 vectors (Monde et al. 2000a; Staub and Maliga 1994; Wostrikoff and Stern 2007). The new synthetic vector backbone in plasmid pSS22 may be used to develop custom expression vectors targeting insertions in the rbcL-accD intergenic region. Derivatives pSS24 and pSS30 carry a c-Myc tagged spectinomycin
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resistance (aadA) marker gene that may be excised with the CRE site-specific recombinase from the plastid genome of plants transformed with vector pSS30. Plastid vectors with synthetic targeting regions such as those reported here will eliminate the need for tedious cloning steps. Synthetic sequences may be used to target any protein-coding region where restriction sites can be removed by silent mutations based on choosing alternative codons. Applications of synthetic vectors will include engineering of coding regions between conveniently located restriction sites in the targeting region of the transformation vector. If the number of codon changes is large, codon usage frequencies should be considered when designing the synthetic DNA. Off limits to re-engineering are regions where the DNA or RNA sequence is important. Examples for regions where only limited changes can be made are regulatory regions involved in transcription initiation and termination, mRNA processing and translation signals. Off limits to re-engineering are structural RNAs, such as the plastid ribosomal RNA subunits and tRNAs. However, structural RNAs are encoded in a relatively small fraction of the plastid genome. For example, the 155, 943 bp tobacco plastid genome consists of 105,259 bp unique sequence, comprising the 86,686 bp Large and 18,573 bp small Single Copy (LSC and SSC) regions and Inverted Repeat regions A and B (IRA, IRB) each 25,342 bp in size (Yukawa et al. 2006; GenBank Accession no. z00044). Excluded form sequence modification in the LSC and SSC regions are the 23 tRNA genes encoded in 2,054 bp of DNA, less than 2% of the single copy region. In the IR region 5,041 bp encode the four ribosomal RNA genes and seven tRNAs, or 19.9% of the IR sequence. Thus, most of the plastid genome is open to targeting by synthetic vectors. Acknowledgments Sugey Ramona Sinagawa-Garcı´a was supported by a Ph.D. scholarship from Consejo Nacional de Ciencia y Tecnologı´a (CONACYT), Me´xico.
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