Marker rescue from the Nicotiana tabacum plastid genome using a plastid/Escherichia coli shuttle vector

We recently reported an 868-bp plastid DNA minicircle, NICE1, that formed during transformation in a transplastomic Nicotiana tabacum line. Shuttle plasmids containing NICEI sequences were maintained extrachromosomally in plastids and shown to undergo recombination with NICE1 sequences on the plastid genome. To prove the general utility of the shuttle plasmids, we tested whether plastid genes outside the NICE1 region could be rescued in Escherichia coli. The NICE1-based rescue plasmid, pNICER1, carries NICE1 sequences for maintenance in plastids, the CoIE1 ori for maintenance in E. coli and a spectinomcyin resistance gene (aadA) for selection in both systems. In addition, pNICERl carries a defective kanamycin resistance gene, kan*, to target the rescue of a functional kanamycin resistance gene, kan, from the recipient plastid genome. pNICERl was introduced into plastids where recombination could occur between the homologous kan/kan* sequences, and subsequently rescued in E. coli to recover the products of recombination. Based on the expression of kanamycin resistance in E. coli and the analysis of three restriction fragment polymorphisms, recombinant kan genes were recovered at a high frequency. Efficient rescue of kan from the plastid genome in E. coli indicates that NICE 1-based plasmids are suitable for rescuing mutations from any part of the plastid genome, expanding the repertoire of genetic tools available for plastid biology.     

Kanamycin resistance as a selectable marker for plastid transformation in tobacco

We report on a novel chimeric gene that confers kanamycin resistance on tobacco plastids. The kan gene from the bacterial transposon Tn5, encoding neomycin phosphotransferase (NPTII), was placed under control of plastid expression signals and cloned between rbcL and ORF512 plastid gene sequences to target the insertion of the chimeric gene into the plastid genome. Transforming plasmid pTNH32 DNA was introduced into tobacco leaves by the biolistic procedure, and plastid transformants were selected by their resistance to 50 g/ml of kanamycin monosulfate. The regenerated plants uniformly transmitted the transplastome to the maternal progeny. Resistant clones resulting from incorporation of the chimeric gene into the nuclear genome were also obtained. However, most of these could be eliminated by screening for resistance to high levels of kanamycin (500 g/ml). Incorporation of kan into the plastid genome led to its amplification to a high copy number, about 10000 per leaf cell, and accumulation of NPTII to about 1% of total cellular protein.     

Next generation synthetic vectors for transformation of the plastid genome of higher plants

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. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A novel approach to plastid transformation utilizes the phiC31 phage integrase

Thus far plastid transformation in higher plants has been based on incorporation of foreign DNA in the plastid genome by the plastid's homologous recombination machinery. We report here an alternative approach that relies on integration of foreign DNA by the phiC31 phage site-specific integrase (INT) mediating recombination between bacterial and phage attachment sites (attB and attP, respectively). Plastid transformation by the new approach depends on the availability of a recipient line in which an attB site has been incorporated in the plastid genome by homologous recombination. Plastid transformation involves insertion of an attP vector into the attB site by INT and selection of transplastomic clones by selection for antibiotic resistance carried in the attP plastid vector. INT function was provided by either expression from a nuclear gene, which encoded a plastid-targeted INT, or expressing INT transiently from a non-integrating plasmid in plastids. Transformation was successful with both approaches using attP vectors with kanamycin resistance or spectinomycin resistance as the selective marker. Transformation efficiency in some of the stable nuclear INT lines was as high as 17 independently transformed lines per bombarded sample. As this system does not rely on the plastid's homologous recombination machinery, we expect that INT-based vectors will make plastid transformation a routine in species in which homologous recombination rarely yields transplastomic clones.     

A plasmid system to monitor gene conversion and reciprocal recombination in vitro

A plasmid system allowing for the detection of recombinagenic activitues in cell-free extracts is described. Two truncated alleles of the bacterial neomycin resistance gene (neo), differing from each other at a polymorphic restriction site, were constructed. Recombinations involving both alleles mediated by Drosophila embryo nuclear protein extracts or Drosophila larva whole cell protein extracts were selected by their ability to confer kanamycin resistance to E. coli. Restriction analysis of plasmids recovered from E. coli transformants allowed the monitoring of the two molecular mechanisms which can lead to functional neo genes, gene conversion and reciprocal recombination.A dose dependent increase in the recombination frequency with increasing amounts of cell extract was observed. Recombination was further increased by linearizing one of the two substrate plasmids. The Drosophila cell extracts catalyzed recombination in vitro since after incubation a recombination product could be identified by polymerase chain reaction (PCR) technology. The recombination was absolutely dependent on the presence of an active cell extract, since no diagnostic PCR product was detected in a reaction where extract was omitted. Analysis of a representative number of recombinant plasmids by restriction analysis revealed that in the absence of an exogenous recombinational system less than 2% of kanamycin resistant recombinant plasmids occurred by gene conversion upon transformation into E. coli. In contrast, recombinants exhibiting restriction patterns diagnostic for gene conversion were observed at frequencies between 5.1% and 9.8% after incubation with Drosophila larva cell extracts. These results strongly argued that gene conversion is a prominent mechanism of recombination in Drosophila mitotic cells.     

Identification of functional lox sites in the plastid genome

Our objective was to test whether or not cyclization recombination (CRE), the P1 phage site-specific recombinase, induces genome rearrangements in plastids. Testing was carried out in tobacco plants in which a DNA sequence, located between two inversely oriented locus of X-over of P1 (loxP) sites, underwent repeated cycles of inversions as a means of monitoring CRE activity. We report here that CRE mediates deletions between loxP sites and plastid DNA sequences in the 3'rps12 gene leader (lox-rps12) or in the psbA promoter core (lox-psbA). We also observed deletions between two directly oriented lox-psbA sites, but not between lox-rps12 sites. Deletion via duplicated rRNA operon promoter (Prrn) sequences was also frequent in CRE-active plants. However, CRE-mediated recombination is probably not directly involved, as no recombination junction between loxP and Prrn could be observed. Tobacco plants carrying deleted genomes as a minor fraction of the plastid genome population were fertile and phenotypically normal, suggesting that the absence of deleted genome segments was compensated by gene expression from wild-type copies. The deleted plastid genomes disappeared in the seed progeny lacking CRE. Observed plastid genome rearrangements are specific to engineered plastid genomes, which contain at least one loxP site or duplicated psbA promoter sequences. The wild-type plastid genome is expected to be stable, even if CRE is present in the plastid.     

Homeologous plastid DNA transformation in tobacco is mediated by multiple recombination events.

Efficient plastid transformation has been achieved in Nicotiana tabacum using cloned plastid DNA of Solanum nigrum carrying mutations conferring spectinomycin and streptomycin resistance. The use of the incompletely homologous (homeologous) Solanum plastid DNA as donor resulted in a Nicotiana plastid transformation frequency comparable with that of other experiments where completely homologous plastid DNA was introduced. Physical mapping and nucleotide sequence analysis of the targeted plastid DNA region in the transformants demonstrated efficient site-specific integration of the 7.8-kb Solanum plastid DNA and the exclusion of the vector DNA. The integration of the cloned Solanum plastid DNA into the Nicotiana plastid genome involved multiple recombination events as revealed by the presence of discontinuous tracts of Solanum-specific sequences that were interspersed between Nicotiana-specific markers. Marked position effects resulted in very frequent cointegration of the nonselected peripheral donor markers located adjacent to the vector DNA. Data presented here on the efficiency and features of homeologous plastid DNA recombination are consistent with the existence of an active RecA-mediated, but a diminished mismatch, recombination/repair system in higher-plant plastids.     

Retrofitting ampicillin resistant vectors by recombination for use in generating C. elegans transgenic animals by bombardment

Caenorhabditis elegans is an important model organism for modern biologic research. An essential aspect of C. elegans research is the production of transgenic animals for study. These are often generated via microinjection, but biolistic bombardment has become increasingly popular. However, many of the plasmids previously generated for use in microinjection are not readily used for bombardment due to the lack of a convenient marker. The unc-119 gene is often used as a marker since unc-119 rescue can be observed at low magnification, allowing rescued animals to be easily distinguished from the larger number of non-rescued animals. Here we report the use of homologous recombination in Escherichia coli as a method to insert a cassette containing the unc-119 gene into commonly used plasmids at the site of the ampicillin resistance gene which is simpler than other methods like subcloning. These cassettes are flanked by regions homologous to the 5′ and 3′ ends of the ampicillin resistance gene and contain either the unc-119 gene and the kanamycin resistance gene or a unc-119:mCherry fusion gene and the kanamycin resistance gene. The resulting plasmids may be used for biolistic bombardment to yield animals that display unc-119 rescue, and also express the recipient plasmid transgene.     

Molecular cloning and mapping of a deletion derivative of the plasmid Rts 1

The plasmid pTW20 is a deletion derivative of the kanamycin resistance plasmid Rts1. By digesting pTW20 DNA with EcoRI endonuclease six fragments were generated, and each was cloned in the vector plasmid pACYC184. These cloned EcoRI fragments were further digested with various endonucleases, and the cleavage map of pTW20 was constructed. A SalI fragment (1.5 Md) in E1 (the largest EcoRI fragment; 11.5 Md) contained the genes kan (kanamycin resistance) and puv (uv sensitization of host). An electron microscopy study of a BamHI fragment containing kan revealed the presence of a transposon-like structure in the fragment. The smallest EcoRI fragment E6 (2.0 Md) was capable of autonomous replication in a polA host, indicating that E6 contained replication genes of pTW20. These genes were found to be located on a 1.1-Md HindIII fragment in E6. Two incompatibility genes were identified on the pTW20 genome, one located on each of the fragments E6 and E5 (3.5 Md), and expressed T incompatibility independently. The nature of the temperature sensitivity of pTW20 was discussed.     

Tobacco plastid ribosomal protein S18 is essential for cell survival

Plastid genomes contain a conserved set of genes most of which are involved in either photosynthesis or gene expression. Among the ribosomal protein genes present in higher plant plastid genomes, rps18 is special in that it is absent from the plastid genomes of several non-green unicellular organisms, including Euglena longa and Toxoplasma gondii. Here we have tested whether the ribosomal protein S18 is required for translation by deleting the rps18 gene from the tobacco plastid genome. We report that, while deletion of the rps18 gene was readily obtained, no homoplasmic Δrps18 plants or leaf sectors could be isolated. Instead, segregation into homoplasmy led to severe defects in leaf development suggesting that the knockout of rps18 is lethal and the S18 protein is required for cell survival. Our data demonstrate that S18 is indispensable for plastid ribosome function in tobacco and support an essential role for plastid translation in plant development. Moreover, we demonstrate the occurrence of flip-flop recombination on short inverted repeat sequences which generates different isoforms of the transformed plastid genome that differ in the orientation a 70 kb segment in the large single-copy region. However, infrequent occurrence of flip-flop recombination and random segregation of plastid genomes result in the predominant presence of only one of the isoforms in many tissue samples. Implications for the interpretation of chloroplast transformation experiments and vector design are discussed.     

Streptomycin and lincomycin resistances are selective plastid markers in cultured Nicotiana cells

Summary Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.     

Generation of marker-free plastid transformants using a transiently cointegrated selection gene

Genetic engineering of higher plant plastids typically involves stable introduction of antibiotic resistance genes as selection markers. Even though chloroplast genes are maternally inherited in most crops, the possibility of marker transfer to wild relatives or microorganisms cannot be completely excluded. Furthermore, marker expression can be a substantial metabolic drain. Therefore, efficient methods for complete marker removal from plastid transformants are necessary. One method to remove the selection gene from higher plant plastids is based on loop-out recombination, a process difficult to control because selection of homoplastomic transformants is unpredictable. Another method uses the CRE/lox system, but requires additional retransformation and sexual crossing for introduction and subsequent removal of the CRE recombinase. Here we describe the generation of marker-free chloroplast transformants in tobacco using the reconstitution of wild-type pigmentation in combination with plastid transformation vectors, which prevent stable integration of the kanamycin selection marker. One benefit of a procedure using mutants is that marker-free plastid transformants can be produced directly in the first generation (T0) without retransformation or crossing.     

Construction and Complementation of In-Frame Deletions of the Essential Escherichia coli Thymidylate Kinase Gene

This work reports the construction of Escherichia coli in-frame deletion strains of tmk, which encodes thymidylate kinase, Tmk. The tmk gene is located at the third position of a putative five-gene operon at 24.9 min on the E. coli chromosome, which comprises the genes pabC, yceG, tmk, holB, and ycfH. To avoid potential polar effects on downstream genes of the operon, as well as recombination with plasmid-encoded tmk, the tmk gene was replaced by the kanamycin resistance gene kka1, encoding amino glycoside 3′-phosphotransferase kanamycin kinase. The kanamycin resistance gene is expressed under the control of the natural promoter(s) of the putative operon. The E. coli tmk gene is essential under any conditions tested. To show functional complementation in bacteria, the E. coli tmk gene was replaced by thymidylate kinases of bacteriophage T4 gp1, E. coli tmk, Saccharomyces cerevisiae cdc8, or the Homo sapiens homologue, dTYMK. Growth of these transgenic E. coli strains is completely dependent on thymidylate kinase activities of various origin expressed from plasmids. The substitution constructs show no polar effects on the downstream genes holB and ycfH with respect to cell viability. The presented transgenic bacteria could be of interest for testing of thymidylate kinase-specific phosphorylation of nucleoside analogues that are used in therapies against cancer and infectious diseases.     

Instability of Plastid DNA in the Nuclear Genome

Functional gene transfer from the plastid (chloroplast) and mitochondrial genomes to the nucleus has been an important driving force in eukaryotic evolution. Non-functional DNA transfer is far more frequent, and the frequency of such transfers from the plastid to the nucleus has been determined experimentally in tobacco using transplastomic lines containing, in their plastid genome, a kanamycin resistance gene (neo) readymade for nuclear expression. Contrary to expectations, non-Mendelian segregation of the kanamycin resistance phenotype is seen in progeny of some lines in which neo has been transferred to the nuclear genome. Here, we provide a detailed analysis of the instability of kanamycin resistance in nine of these lines, and we show that it is due to deletion of neo. Four lines showed instability with variation between progeny derived from different areas of the same plant, suggesting a loss of neo during somatic cell division. One line showed a consistent reduction in the proportion of kanamycin-resistant progeny, suggesting a loss of neo during meiosis, and the remaining four lines were relatively stable. To avoid genomic enlargement, the high frequency of plastid DNA integration into the nuclear genome necessitates a counterbalancing removal process. This is the first demonstration of such loss involving a high proportion of recent nuclear integrants. We propose that insertion, deletion, and rearrangement of plastid sequences in the nuclear genome are important evolutionary processes in the generation of novel nuclear genes. This work is also relevant in the context of transgenic plant research and crop production, because similar processes to those described here may be involved in the loss of plant transgenes.     

Extensive homologous recombination between introduced and native regulatory plastid DNA elements in transplastomic plants

Homologous recombination within plastids directs plastid genome transformation for foreign gene expression and study of plastid gene function. Though transgenes are generally efficiently targeted to their desired insertion site, unintended homologous recombination events have been observed during plastid transformation. To understand the nature and abundance of these recombination events, we analyzed transplastomic tobacco lines derived from three different plastid transformation vectors utilizing two different loci for foreign gene insertion. Two unintended recombinant plastid DNA species were formed from each regulatory plastid DNA element included in the transformation vector. Some of these recombinant DNA species accumulated to as much as 10–60% of the amount of the desired integrated transgenic sequence in T0 plants. Some of the recombinant DNA species undergo further, “secondary” recombination events, resulting in an even greater number of recombinant plastid DNA species. The abundance of novel recombinant DNA species was higher in T0 plants than in T1 progeny, indicating that the ancillary recombination events described here may have the greatest impact during selection and regeneration of transformants. A line of transplastomic tobacco was identified containing an antibiotic resistance gene unlinked from the intended transgene insertion as a result of an unintended recombination event, indicating that the homologous recombination events described here may hinder efficient recovery of plastid transformants containing the desired transgene. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genome evolution of a tertiary dinoflagellate plastid

The dinoflagellates have repeatedly replaced their ancestral peridinin-plastid by plastids derived from a variety of algal lineages ranging from green algae to diatoms. Here, we have characterized the genome of a dinoflagellate plastid of tertiary origin in order to understand the evolutionary processes that have shaped the organelle since it was acquired as a symbiont cell. To address this, the genome of the haptophyte-derived plastid in Karlodinium veneficum was analyzed by Sanger sequencing of library clones and 454 pyrosequencing of plastid enriched DNA fractions. The sequences were assembled into a single contig of 143 kb, encoding 70 proteins, 3 rRNAs and a nearly full set of tRNAs. Comparative genomics revealed massive rearrangements and gene losses compared to the haptophyte plastid; only a small fraction of the gene clusters usually found in haptophytes as well as other types of plastids are present in K. veneficum. Despite the reduced number of genes, the K. veneficum plastid genome has retained a large size due to expanded intergenic regions. Some of the plastid genes are highly diverged and may be pseudogenes or subject to RNA editing. Gene losses and rearrangements are also features of the genomes of the peridinin-containing plastids, apicomplexa and Chromera, suggesting that the evolutionary processes that once shaped these plastids have occurred at multiple independent occasions over the history of the Alveolata.     

Genetic engineering of Treponema pallidum subsp. pallidum,the Syphilis Spirochete

Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kan^R) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kan^R gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kan^R cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kan^R cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl₂-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kan^R cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kan^R message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprA^ko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.     

In vivo testing of a tobacco plastid DNA segment for guide RNA function in psbL editing

A C-to-U RNA editing event creates a functional initiation codon for translation of the psbL mRNA in tobacco plastids. Small trans-acting guide RNAs (gRNAs) have been shown to be involved in editing site selection in kinetoplastid mitochondria. A computer search of the tobacco plastid genome (ptDNA) identified such a putative gRNA, a 14-nucleotide sequence motif that is complementary to the psbL mRNA, including the A nucleotide required to direct the C-to-U change. The critical A nucleotide of the putative gRNA gene was changed to G by plastid transformation. We report here that the introduced mutation did not abolish psbL editing. Since no other region of the plastid genome contains significant complementarity to the psbL editing site we suggest that, if gRNAs serve as trans-acting factors for plastid psbL mRNA editing, they either have only a limited complementarity to the editing site, or are encoded in the nuclear genome.