Plastid Transformation System Research Articles

Plastid Engineering of a Marine Alga, Nannochloropsis gaditana, for Co-Expression of Two Recombinant Peptides.

The purpose of this study was to establish a plastid transformation system for expressing recombinant proteins in Nannochloropsis gaditana. On the basis of the sequenced plastid genome, the homologous flanking region, 16S-trnI/trnA-23S, and the endogenous regulatory fragments containing the psbA promoter, rbcL promoter, rbcL terminator, and psbA terminator were amplified from N. gaditana as elements of a plastid transformation vector. Then, the herbicide-resistant gene (bar) was used as a selectable marker, regulated by the psbA promoter and rbcL terminator. Finally, two codon-optimized antimicrobial peptide-coding genes linked by endogenous ribosome binding site (RBS) in a polycistron were inserted into the constructed vector under the regulation of the rbcL promoter and psbA terminator. After microparticle bombardment, the positive clones were detected using polymerase chain reaction (PCR), and Southern and Western blotting were used to assess the co-expression of the two antimicrobial peptides from the plastid. Nannochloropsis gaditana showed the potential to express recombinant proteins for biotechnological applications, for example, for the development of oral vaccines in aquaculture.

Plastid Transformation in Poplar: A Model for Perennial Trees.

Poplar (Populus) is an important forest tree and considered model for perennial trees. Here we describe a method for poplar plastid transformation, which involves preparation of explants, vector construction, biolistic bombardment, regeneration and selection of transplastomic poplar plants. The young leaves of 4-week-old poplar plants are used for biolistic bombardment and aadA gene as selectable marker. Homoplasmic transplastomic lines are obtained after regeneration and several rounds of selection with spectinomycin over 10months. Homoplasmy is further confirmed by Southern blot. The establishment of a plastid transformation system for Populus is likely to make a significant contribution to tree genetic improvement.

Plastid genome engineering: novel optimization strategies and applications

The plastid genome engineering system allows site-specific modifications via two homologous recombination events. It is much safer, more precise and efficient compared with the nuclear transformation system. This technology can be applied to the basic research to expand plastid genome function analysis, and it also provides an excellent platform for not only high-level production of recombinant proteins but also plant breeding. In this review, we summarize the state of the art and progresses in this field. We focus on novel breeding strategies in transformation system improvement and new tools to enhance plastid transgene expression levels. In addition, we highlight selected applications in resistance engineering and quality improvement via metabolic engineering. We believe that by overcoming current technological limitations in the plastid transformation system can another green revolution for crop breeding beckon.

Modifying fatty acid profiles through a new cytokinin-based plastid transformation system.

The widespread use of herbicides and antibiotics for selection of transgenic plants has not been very successful with regard to commercialization and public acceptance. Hence, alternative selection systems are required. In this study, we describe the use of ipt, the bacterial gene encoding the enzyme isopentenyl transferase from Agrobacterium tumefaciens, as a positive selectable marker for plastid transformation. A comparison between the traditional spectinomycin-based aadA selection system and the ipt selection system demonstrated that selection of transplastomic plants on medium lacking cytokinin was as effective as selection on medium containing spectinomycin. Proof of principle was demonstrated by transformation of the kasIII gene encoding 3-ketoacyl acyl carrier protein synthaseIII into tobacco plastids. Transplastomic tobacco plants were readily obtained using the ipt selection system, and were phenotypically normal despite over-expression of isopentenyl transferase. Over-expression of KASIII resulted in a significant increase in 16:0 fatty acid levels, and a significant decrease in the levels of 18:0 and 18:1 fatty acids. Our study demonstrates use of a novel positive plastid transformation system that may be used for selection of transplastomic plants without affecting the expression of transgenes within the integrated vector cassette or the resulting activity of the encoded protein. This system has the potential to be applied to monocots, which are typically not amenable to traditional antibiotic-based selection systems, and may be used in combination with a negative selectable marker as part of a two-step selection system to obtain homoplasmic plant lines.

ChlB Requirement for Chlorophyll Biosynthesis under Short Photoperiod in Marchantia polymorpha L.

Chlorophylls (Chls) play pivotal roles in energy absorption and transduction and also in charge separation in reaction centers in all photosynthetic organisms. In Chl biosynthesis steps, only a step for the enzymatic reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) is mediated by both nuclear- and chloroplast-encoded genes in land plants. Many plants encode the genes for light-dependent Pchlide reductase (LPOR) and light-independent Pchlide reductase (DPOR) in the nucleus and chloroplast genome, respectively. During the diversification of land plants, the reduction step of Pchlide to Chlide has become solely dependent on LPOR, and the genes for DPOR have been lost from chloroplast genome. It remains unclear why DPOR persists in some land plants, how they were eliminated from chloroplast genomes during the diversification of land plants, and under what environmental conditions DPOR was required. We demonstrate that DPOR is functional in liverwort (Marchantia polymorpha L.) and plays an important role in Chl biosynthesis. Having established a plastid transformation system in liverwort, we disrupted chlB, which encodes a subunit of DPOR in the M. polymorpha chloroplast genome. Morphological and Chl content analysis of a chlB mutant grown under different photoperiods revealed that DPOR is particularly required for Chl biosynthesis under short-day conditions. Our findings suggest that an environmental condition in the form of photoperiod is an important factor that determines the loss or retention of chloroplast-encoded genes mediating Pchlide reduction to Chlide.

Dengue virus tetra-epitope peptide expressed in lettuce chloroplasts for potential use in dengue diagnosis.

Dengue virus causes about 100 million cases of dengue disease per year in the world. Laboratory diagnosis is done mainly by serological techniques, which in many cases use crude virus extracts that may cause cross-reactions to other flaviviruses. These undesirable cross-reactions can be reduced or eliminated by using recombinant proteins based on restricted epitopes. Aiming to decrease flaviviral cross-reactions and non-specific interactions in dengue serological assays, a plant expression system was chosen for recombinant antigen production as a reliable and inexpensive dengue diagnostic tool. In the present report, the lettuce plastid transformation system was applied to achieve efficient and stable tetra-epitope peptide antigen production, and its reactivity was evaluated. For this purpose, one putative epitope at positions 34 to 57 of E protein within the junction site of domains I and II of dengue virus (DENV) 1 to 4 serotypes linked by glycine linkers was expressed in lettuce chloroplasts. The potential immunoreactivity for the four DENV serotypes was evaluated using sera from patients of positive and negative dengue cases. Results indicated an overall sensitivity of 71.7% and specificity of 100%. No cross-reactions with the sera of yellow fever-positive or healthy individuals vaccinated against yellow fever were observed. This novel approach may provide an alternative system for the large-scale production of dengue recombinant antigens useful for serodiagnosis.

The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA.

BackgroundDunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri.ResultsThe D. salina organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA) sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA.ConclusionsThese findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the development of a viable plastid transformation system for this model alga, and they will complement the forthcoming D. salina nuclear genome sequence, placing D. salina in a group of a select few photosynthetic eukaryotes for which complete genome sequences from all three genetic compartments are available.

Stable Plastid Transformation in Nicotiana benthamiana

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.

Simple and efficient plastid transformation system for the liverwort Marchantia polymorpha 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.

Transformation of poplar (Populus alba) plastids and expression of foreign proteins in tree chloroplasts

Plastid transformation offers several unique advantages compared with nuclear genome transformation, such as high level of transgene expression within plastids, expressing multiple transgenes as operons, lack of position effect due to site-specific transgene integration, and reducing risks of gene flow via pollen due to maternal inheritance of the plastid genome. Plastid transformation has been applied to several herbal species, but as yet there are no applications to tree species. We report here the first successful plastid transformation in a tree species, Populus alba. A vector for plastid transformation of poplar (Populus alba) was constructed, which carried the spectinomycin resistance gene and the green fluorescence protein gene as marker genes. In the regenerated shoots, the site-specific integration of foreign genes and the establishment of a high homoplastomic state were confirmed. Immunoblot analysis and histological observations corroborated the accumulation of green fluorescence protein in chloroplasts. The establishment of a plastid transformation system in poplar provides a novel tool for tree biotechnology.

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 approximately 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.

Stable plastid transformation in lettuce (Lactuca sativa L.).

Although plastid transformation in higher plants was first demonstrated in the early 1990s it is only recently that the technology is being extended to a broader range of species. To date, the production of fertile transplastomic plants has been reported for tobacco, tomato, petunia, soybean, cotton and Lesquerella fendleri (Brassicaceae). In this study we demonstrate a polyethylene glycol-mediated plastid transformation system for lettuce that generates fertile, homoplasmic, plastid-transformed lines. Transformation was achieved using a vector that targets genes to the trnA/trnI intergenic region of the lettuce plastid genome employing the aadA gene as a selectable marker against spectinomycin. Spectinomycin resistance and heterologous gene transcription were shown in T(1) plants derived from self-pollinated primary regenerants demonstrating transmission of the plastid-encoded transgene to the first seed generation. Crossing with male sterile wild-type lettuce showed that spectinomycin resistance was not transmitted via pollen. Constructs containing the gfp gene showed plastid-based expression of green fluorescent protein. The lettuce plastid could have potential both as a production and a delivery system for edible human therapeutic proteins.

Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit.

Transgenic chloroplasts offer unique advantages in plant biotechnology, including high-level foreign protein expression, absence of epigenetic effects, and gene containment due to the lack of transgene transmission through pollen. However, broad application of plastid genome engineering in biotechnology has been largely hampered by both the lack of chloroplast transformation systems for major crop plants and the usually low plastid gene expression levels in nongreen tissues such as fruits, tubers, and other storage organs. Here we describe the development of a plastid transformation system for tomato, Lycopersicon esculentum. This is the first report on the generation of fertile transplastomic plants in a food crop with an edible fruit. We show that chromoplasts in the tomato fruit express the transgene to approximately 50% of the expression levels in leaf chloroplasts. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts (>40% of the total soluble protein), this system paves the way to efficient production of edible vaccines, pharmaceuticals, and antibodies in tomato.

Technical Advance: 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.