Plastid marker gene excision by the phiC31 phage site-specific recombinase

Marker genes are essential for selective amplification of rare transformed plastid genome copies to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here we report excision of plastid marker genes by the phiC31 phage site-specific integrase (Int) that mediates recombination between bacterial (attB) and phage (attP) attachment sites. We tested marker gene excision in a two-step process. First we transformed the tobacco plastid genome with the pCK2 vector in which the spectinomycin resistance (aadA) marker gene is flanked with suitably oriented attB and attP sites. The transformed plastid genomes were stable in the absence of Int. We then transformed the nucleus with a gene encoding a plastid-targeted Int that led to efficient marker gene excision. The aadA marker free Nt-pCK2-Int plants were resistant to phosphinothricin herbicides since the pCK2 plastid vector also carried a bar herbicide resistance gene that, due to the choice of its promoter, causes a yellowish-golden (aurea) phenotype. Int-mediated marker excision reported here is an alternative to the currently used CRE/loxP plastid marker excision system and expands the repertoire of the tools available for the manipulation of the plastid genome.     

Plastid transformation in eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

We have developed a method for plastid transformation in eggplant (Solanum melongena L.), a solanaceous plant species. Plastid transformation in eggplant was achieved by bombardment of green stem segments with pPRV111A plastid expression vector carrying the aadA gene encoding aminoglycoside 3′′-adenylyltransferase. Biolistic delivery of the pPRV111A plasmid yielded transplastomic plants at a frequency of two per 21 bombarded plates containing 25 stem explants each. Integration of the aadA gene in the plastome was verified by PCR analysis and also by Southern blotting using 16S rDNA (targeting sequence) and the aadA gene as a probe. Transplastomic expression of the aadA gene was verified by RT-PCR. The development of transplastomic technology in eggplant may open up exciting possibilities for novel gene introduction and expression in the engineered plastome for agronomic or pharmaceutical traits.     

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.     

Plastid transformation in Arabidopsis thaliana

Plastid transformation is reported in Arabidopsis thaliana following biolistic delivery of transforming DNA into leaf cells. Transforming plasmid pGS31A carries a spectinomycin resistance (aadA) gene flanked by plastid DNA sequences to target its insertion between trnV and the rps12/7 operon. Integration of aadA by two homologous recombination events via the flanking ptDNA sequences and selective amplification of the transplastomes on spectinomycin medium yielded resistant cell lines and regenerated plants in which the plastid genome copies have been uniformly altered. The efficiency of plastid transformation was low: 2 in 201 bombarded leaf samples. None of the 98 plants regenerated from the two lines were fertile. Received: 13 February 1998 / Revision received: 24 April 1998 / Accepted: 5 June 1998     

Gene activation in plastids by the CRE site-specific recombinase

We developed a novel system for gene activation in plastids that uses the CRE/loxP site-specific recombination system to create a translatable reading frame by excision of a blocking sequence. To test the system, we introduced an inactive gfp* gene into the tobacco plastid genome downstream of the selectable spectinomcyin resistance (aadA) marker gene. The aadA gene is the blocking sequence, and is flanked by directly oriented loxP sites for excision by the CRE. In the non-activated state, gfp* is transcribed from the aadA promoter, but the mRNA is not translated due to the lack of an AUG translation initiation codon. Green Fluorescent Protein (GFP) expression is activated by excision of the aadA coding segment to link up the gfp* coding region with the translation initiation codon of aadA. Tobacco plants that carry the inactive gfp* gene do not contain detectable levels of GFP. However, activation of gfp* resulted in GFP accumulation, proving the utility of CRE-induced protein expression in tobacco chloroplasts. The gene activation system described here will be useful to probe plastid gene function and for the production of recombinant proteins in chloroplasts.     

Simple and efficient plastid transformation system for the liverwort <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> L. suspension-culture cells

We have established a simple and efficient plastid transformation system for liverwort, Marchantia polymorpha L., suspension-culture cells, which are homogenous, chloroplast-rich and␣rapidly growing. Plasmid pCS31 was constructed to integrate an aadA expression cassette for spectinomycin-resistance into the trnI–trnA intergenic region of the liverwort plastid DNA by homologous recombination. Liverwort suspension-culture cells were bombarded with pCS31-coated gold projectiles and selected on a medium containing spectinomycin. Plastid transformants were reproducibly isolated from the obtained spectinomycin-resistant calli. Selection on a sucrose-free medium greatly improved the efficiency of selection of plastid transformants. Homoplasmic plastid transformant lines were established by␣successive subculturing for 14 weeks or longer on the spectinomycin-containing medium. The plastid transformation system of liverwort suspension-culture cells should facilitate the investigation of the fundamental genetic systems of plastid DNA, such as replication.     

Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker

We describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco-specific plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/Trps16::PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf-based regenera- tion system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastid-expressed green fluorescent protein was used as a visual marker for identification of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.     

Stable plastid transformation in <Emphasis Type="Italic">Scoparia dulcis</Emphasis> L.

In the present investigation we report stable plastid transformation in Scoparia dulcis L., a versatile medicinal herb via particle gun method. The vector KNTc, harbouring aadA as a selectable marker and egfp as a reporter gene which were under the control of synthetic promoter pNG1014a, targets inverted repeats, trnR/t rnN of the plastid genome. By use of this heterologous vector, recovery of transplastomic lines with suitable selection protocol have been successfully established with overall efficiency of two transgenic lines for 25 bombarded leaf explants. PCR and Southern blot analysis demonstrated stable integration of foreign gene into the target sequences. The results represent a significant advancement of the plastid transformation technology in medicinal plants, which relevantly implements a change over in enhancing and regulating of certain metabolic pathways.     

Stable Plastid Transformation in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Plastids from Nicotiana benthamiana were transformed with the vector for dicistronic expression of two genes—aminoglycoside 3'-adenyltransferase (aadA) and green fluorescent protein (gfp)—in the plastids of Nicotiana tabacum. Transplastomic shoots exhibited green fluorescence under UV light. Transformation efficiencies were similar between species. Although the border sequence (trnI and trnA) for homologous recombination to transform the plastid genome of N. benthamiana was identical to that sequence of N. tabacum, the exception was a 9-bp addition in the intron of trnI. This indicated that the N. tabacum sequence used as a border region for recombination was sufficient to insert the foreign gene into the target site between the trnI and trnA of N. benthamiana with similar efficiency. Southern blot analysis detected the presence of aadA and gfp between trnI and trnA in the plastid genome of N. benthamiana. Northern and western blot analyses revealed high expression of gfp in the plastids from petals and leaves. Our results suggest that the plastid transformation system established here is applicable to investigations of the interactions between plastid and nucleus in N. benthamiana.     

Efficient and Stable Transformation of Lactuca sativa L. cv. Cisco (lettuce) Plastids

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to ~36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.     

The maize CorA/MRS2/MGT-type Mg transporter,ZmMGT10, responses to magnesium deficiency and confers low magnesium tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Key message

A new selectable marker gene for stable transformation of the plastid genome was developed that is similarly efficient as the aadA, and produces no background of spontaneous resistance mutants.

Abstract

More than 25 years after its development for Chlamydomonas and tobacco, the transformation of the chloroplast genome still represents a challenging technology that is available only in a handful of species. The vast majority of chloroplast transformation experiments conducted thus far have relied on a single selectable marker gene, the spectinomycin resistance gene aadA. Although a few alternative markers have been reported, the aadA has remained unrivalled in efficiency and is, therefore, nearly exclusively used. The development of new marker genes for plastid transformation is of crucial importance to all efforts towards extending the species range of the technology as well as to those applications in basic research, biotechnology and synthetic biology that involve the multistep engineering of plastid genomes. Here, we have tested a bifunctional resistance gene for its suitability as a selectable marker for chloroplast transformation. The bacterial enzyme aminoglycoside acetyltransferase(6′)-Ie/aminoglycoside phosphotransferase(2″)-Ia possesses an N-terminal acetyltransferase domain and a C-terminal phosphotransferase domain that can act synergistically and detoxify aminoglycoside antibiotics highly efficiently. We report that, in combination with selection for resistance to the aminoglycoside tobramycin, the aac(6′)-Ie/aph(2″)-Ia gene represents an efficient marker for plastid transformation in that it produces similar numbers of transplastomic lines as the spectinomycin resistance gene aadA. Importantly, no spontaneous antibiotic resistance mutants appear under tobramycin selection.

     

Stability of Soybean Recombinant Plastome Over Six Generations

The stability of a plastid transgene has been evaluated in soybean transformants over six generations. These transformants had integrated the aadA selection cassette in the intergenic region between the rps12/7 and trnV genes. Three independent homoplasmic T0 transformation events were selected and ten plants from each event propagated to generation T5 in the absence of selection pressure. No transgene rearrangement nor wild-type plastome were detected in generation T5 by Southern blot analysis. All tested progenies were uniformly resistant to spectinomycin. Therefore, soybean transformants of generations T0 and T5 appear to be genetically and phenotypically identical.     

Chloroplast Transformation in Oilseed Rape

The chloroplast transformation vector pNRAB carries two expression cassettes for the spectinomycin resistance gene aadA and the insect resistance gene cry1Aa10. The two cassettes are sited between the rps7 and ndhB targeting fragments. Biolistic delivery of the vector DNA, followed by spectinomycin selection, yielded chloroplast transformants at a frequency of four in 1000 bombarded cotyledon petioles. PCR analysis and Southern blot of PCR products confirmed the site-specific integration of aadA and cry1Aa10 into the chloroplast genomes of transgenic oilseed rape. When transgenic oilseed rape leaves were fed to second instar Plutella xylostera larvae, 47% mortality was observed against this insect and the surviving larvae had significantly lower weight than the control. This is the first report of chloroplast transformation in oilseed rape and the introduction of novel genes between the rps7 and ndhB genes in the chloroplast genome. This offers an opportunity for improvement of oilseed rape by chloroplast genetic engineering.     

Selectable marker recycling in the chloroplast

The bacterial geneaadA is an important and widely used selectable marker for manipulation of the chloroplast genome through biolistic transformation. Because no other such marker is available, two strategies for recycling of theaadA cassette have been developed. One utilizes homologous recombination between two direct repeats flanking theaadA cassette to allow its loss under non-selective growth conditions. A second strategy is to perform co-transformation with a plasmid containing a modified, non-essential chloroplast gene and another plasmid in which theaadA cassette disrupts a chloroplast gene known to be essential for survival. Under selective growth conditions the first mutation can be transferred to all chloroplast DNA copies whereas theaadA insertion remains heteroplasmic. Loss of the selectable marker can be achieved subsequently by growing the cells on non-selective media. In both cases it is possible to reuse theaadA cassette for the stepwise disruption or mutagenesis of any gene in the same strain.     

A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1

Here we report an approach to generate a knock-in mouse model using an ‘ends-out’ gene replacement vector to substitute the murine Parp-1 (mParp-1) coding sequence (32 kb) with its human orthologous sequence (46 kb). Unexpectedly, examination of mutant ES cell clones and mice revealed that site-specific homologous recombination was mimicked in three independently generated ES cell clones by bidirectional extension of the vector homology arms using the endogenous mParp-1-flanking sequences as templates. This was followed by adjacent integration of the targeting vector, thus leaving the endogenous mParp-1 locus functional. A related phenomenon termed ‘ectopic gene targeting’ has so far only been described for ‘ends-in’ integration-type vectors in non-ES cell gene targeting. We provide reliable techniques to detect such ectopic gene targeting which represents an unexpected caveat in mouse genetic engineering that should be considered in the design and validation strategy of future gene knock-in approaches. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A disarmed binary vector from Agrobacterium tumefaciens functions in Agrobacterium rhizogenes

Summary Binary Ti plasmid vector systems consist of two plasmids in Agrobacterium, where one plasmid contains the DNA that can be transferred to plant cells and the other contains the virulence (vir) genes which are necessary for the DNA transfer but are not themselves stably transferred. We have constructed two nononcogenic vectors (pARC4 and pARC8) based on the binary Ti plasmid system of Agrobacterium tumefaciens for plant transformation. Each vector contains the left and right termini sequences from pTiT37. These sequences, which determine the extent of DNA transferred to plant cells, flank unique restriction enzyme sites and a marker gene that functions in the plant (nopaline synthase in pARC4 or neomycin phosphotransferase in pARC8). After construction in vitro, the vectors can be conjugatively transferred from E. coli to any of several Agrobacterium strains containing vir genes. Using A. rhizogenes strain A4 containing the resident Ri plasmid plus a vector with the nopaline synthase marker, we found that up to 50% of the hairy roots resulting from the infection of alfalfa or tomato synthesized nopaline. Thus, vector DNA encoding an unselected marker was frequently co-transferred with Ri plasmid DNA to an alfalfa or a tomato cell. In contrast, the frequency of co-transfer to soybean cells was difficult to estimate because we encountered a high background of non-transformed roots using this species. Up to five copies of the vector DNA between the termini sequences were faithfully transferred and maintained in most cases suggesting that the termini sequences and the vir genes from the Ri and Ti plasmids are functionally equivalent.     

A set of modular plant transformation vectors allowing flexible insertion of up to six expression units

We have constructed a binary vector for Agrobacterium-mediated plant transformation, which has a multiple cloning site consisting of 13 hexanucleotide restriction sites, 6 octanucleotide restriction sites and 5 homing endonuclease sites. The homing endonuclease sites have the advantages to be extremely rare in natural sequences and to allow unidirectional cloning. We have also constructed a set of auxiliary vectors allowing the assembly of expression cassettes flanked by homing endonuclease sites. The expression cassettes assembled in these auxiliary vectors can be transferred into the binary vector with virtually no risk of cutting the vector within previously introduced sequences. This vector set is ideally suited for the construction of plant transformation vectors containing multiple expression cassettes and/or other elements such as matrix attachment regions. With this modular vector system, six different expression units were constructed in as many auxiliary vectors and assembled together in one plant transformation vector. The transgenic nature of Arabidopsis thaliana plants, transformed with this plant transformation vector, was assessed and the expression of each of the six genes was demonstrated.     

Precise excision of plastid DNA by the large serine recombinase Bxb1

Marker genes are essential for the selection and identification of rarely occurring transformation events generated in biotechnology. This includes plastid transformation, which requires that multiple copies of the modified chloroplast genome be present to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here, we demonstrate the precise excision of attP‐ and attB‐flanked DNA from the plastid genome mediated by the large serine recombinase Bxb1. We transformed the tobacco plastid genome with the pTCH‐PB vector containing a stuffer fragment of DNA flanked by directly oriented nonhomologous attP and attB recombinase recognition sites. In the absence of the Bxb1 recombinase, the transformed plastid genomes were stable and heritable. Nuclear‐transformed transgenic tobacco plants expressing a plastid‐targeted Bxb1 recombinase were crossed with transplastomic pTCH‐PB plants, and the T₁ hybrids exhibited efficient excision of the target sequence. The Bxb1–att system should prove to be a useful tool for site‐specifically manipulating the plastid genome and generating marker‐free transplastomic plants.