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Plant Physiology 145:1129-1143 (2007) © 2007 American Society of Plant Biologists Chloroplast Vector Systems for Biotechnology Applications1Department of Molecular Biology and Microbiology, College of Medicine, University of Central Florida, Orlando, Florida 32816–2364
Chloroplasts are ideal hosts for expression of transgenes. Transgene integration into the chloroplast genome occurs via homologous recombination of flanking sequences used in chloroplast vectors. Identification of spacer regions to integrate transgenes and endogenous regulatory sequences that support optimal expression is the first step in construction of chloroplast vectors. Thirty-five sequenced crop chloroplast genomes provide this essential information. Various steps involved in the design and construction of chloroplast vectors, DNA delivery, and multiple rounds of selection are described. Several crop species have stably integrated transgenes conferring agronomic traits, including herbicide, insect, and disease resistance, drought and salt tolerance, and phytoremediation. Several crop chloroplast genomes have been transformed via organogenesis (cauliflower [Brassica oleracea], cabbage [Brassica capitata], lettuce [Lactuca sativa], oilseed rape [Brassica napus], petunia [Petunia hybrida], poplar [Populus spp.], potato [Solanum tuberosum], tobacco [Nicotiana tabacum], and tomato [Solanum lycopersicum]) or embryogenesis (carrot [Daucus carota], cotton [Gossypium hirsutum], rice [Oryza sativa], and soybean [Glycine max]), and maternal inheritance of transgenes has been observed. Chloroplast-derived biopharmaceutical proteins, including insulin, interferons (IFNs), and somatotropin (ST), have been evaluated by in vitro studies. Human INF
Chloroplasts are members of a class of organelles known as plastids and are found in plant cells and eukaryotic algae. As the site of photosynthesis, chloroplasts are the primary source of the world's food productivity and they sustain life on this planet. Other important activities that occur in plastids include evolution of oxygen, sequestration of carbon, production of starch, synthesis of amino acids, fatty acids, and pigments, and key aspects of sulfur and nitrogen metabolism. Chloroplasts are generally considered as derivative of a cyanobacterial ancestor that was captured early during the evolution of a eukaryotic cell. However, the chloroplast genome is considerably reduced in size as compared to that of free-living cyanobacteria, but the genomic sequences that are still present show clear similarities (Martin et al., 2002  ). Land plant chloroplast genomes typically contain 110 to 120 unique genes, whereas cyanobacteria contain more than 1,500 genes. Many of the missing genes are present in the nuclear genome of the host (Martin et al., 2002).
In most angiosperm plant species, plastid genes are maternally inherited (Hagemann, 2004
Another advantage of plastid transformation is the ability to accumulate large amounts of foreign protein (up to 46% of total leaf protein) when the transgene is stably integrated (De Cosa et al., 2001
Chloroplast genetic engineering also offers the unique advantage of transgene stacking, i.e. simultaneous expression of multiple transgenes, creating an opportunity to produce multivalent vaccines in a single transformation step. Several heterologous operons have been expressed in transgenic chloroplasts, and polycistrons are translated without processing into monocistrons (Quesada-Vargas et al., 2005
The chloroplast genome typically consists of basic units of double-stranded DNA of 120 to 220 kb arranged in monomeric or multimeric circles as well as in linear molecules (Palmer, 1985 ; Lilly et al., 2001). The chloroplast genome generally has a highly conserved organization (Raubeson and Jansen, 2005), with most land plant genomes having two identical copies of a 20- to 30-kb inverted repeat region (IRA and IRB) separating a large single copy (LSC) region and a small single copy (SSC) region. Plastid transformation is typically based on DNA delivery by the biolistic process (Daniell et al., 1990; Sanford et al., 1993) or occasionally by polyethylene glycol (PEG) treatment of protoplasts (Golds et al., 1993; O'Neill et al., 1993). This is followed by transgene integration into the chloroplast genome via homologous recombination facilitated by a RecA-type (Cerutti et al., 1992) system between the plastid-targeting sequences of the transformation vector and the targeted region of the plastid genome. Chloroplast transformation vectors are thus designed with homologous flanking sequences on either side of the transgene cassette to facilitate double recombination. Targeting sequences have no special properties other than that they are homologous to the chosen target site and are generally about 1 kb in size. Both flanking sequences are essential for homologous recombination. Transformation is accomplished by integration of the transgene into a few genome copies, followed by 25 to 30 cell divisions under selection pressure to eliminate untransformed plastids, thereby achieving a homogeneous population of plastid genomes. If the transgene is targeted into the IR region, integration in one IR is followed by the phenomenon of copy correction that duplicates the introduced transgene into the other IR as well.
Transgenes have been stably integrated at several sites within the plastid genome. Transgenes were first integrated into transcriptionally silent spacer regions (Svab and Maliga, 1993
The proposal of a "universal vector" containing the trnA and trnI genes from the IR region of the tobacco chloroplast genome as flanking sequences for homologous recombination to transform several other plant species (of unknown genome sequence) was suggested several years ago (Daniell et al., 1998 ). This concept was based on the high conservation of this intergenic spacer region among the higher plant chloroplast genomes. Vectors designed for transformation of the tobacco plastid genome have been successfully used for potato and tomato plastid transformation, because the homologous flanking sequences present in these vectors showed adequate homology to the corresponding sequences of potato and tomato plastid DNA but the efficiency of transformation is significantly lower than tobacco (Sidorov et al., 1999; Ruf et al., 2001). For example, only one potato and tomato chloroplast transgenic line was obtained per 35 and 87 bombarded plates, respectively, when compared to about 15 tobacco chloroplast transgenic lines often generated from one bombarded plate (Fernandez-San Millan et al., 2003). A similar lower efficiency was observed when petunia flanking sequences (approximately 98% homologous) were used to transform the tobacco chloroplast genome (DeGray et al., 2001), revealing that a lack of complete homology may reduce the transformation efficiency to a great extent. However, comparison of intergenic spacer regions among members of Solanaceae revealed that only four regions are identical (Daniell et al., 2006). Similarly, comparison of intergenic spacer regions of nine grass chloroplast genomes revealed that not even a single spacer region is identical among all sequenced chloroplast genomes (Saski et al., 2007). Therefore, the concept of a universal vector is applicable when a higher level of homology exists among plant species but will be less efficient than species-specific chloroplast vectors. The accession numbers for several crop chloroplast genome sequences have been provided at the Web site (http://www.bch.umontreal.ca/ogmp/projects/other/cp_list.html, http://www.ncbi.nlm.nih.gov/genomes/static/euk_o.html, or http://chloroplast.cbio.psu.edu/cgi-bin/organism.cgi for access to genomic sequences). Additionally, optimization of transformation protocols specific for each crop should enhance the efficiency of transformation.
At the beginning, selection of plastid transformants was carried out by spectinomycin resistance encoded in the mutant 16S ribosomal RNA (rRNA) gene (Harris et al., 1989 ; Svab et al., 1990). Stable integration and expression of the aadA gene was first reported in the chloroplast genome of Chlamydomonas (Goldschmidt-Clermont, 1991). The aadA gene encodes the enzyme aminoglycoside 3' adenylyltransferase that inactivates spectinomycin and streptomycin by adenylation and prevents binding to chloroplast ribosomes. The aadA gene was later used as a selectable marker in tobacco, and the frequency of transformation events increased to 100-fold more than the mutant 16S rRNA genes (Svab and Maliga, 1993). Due to the recessive nature of the mutant 16S rRNA marker gene, the phenotypic resistance was not expressed until sorting out of the transgenomes was essentially completed. Lack of phenotypic resistance permitted the loss of the resistant rRNA gene in 99 out of 100 potential transformation events. Although it was first explained that spectinomycin offers nonlethal selection (Svab and Maliga, 1993) by not inhibiting cell division and growth at high concentrations (approximately 500 µg mL–1), it was observed to be lethal in all other plant species (Table I
).
The neo gene is another alternative marker for plastid transformation that confers kanamycin resistance (Carrer et al., 1993 ). A different kanamycin resistance gene (aphA6) with relatively high transformation efficiency was reported later (Huang et al., 2002). Another selection strategy utilizing a "double barrel" vector was used for cotton transformation where explant for transformation was nongreen cells (Kumar et al., 2004b). The cotton plastid transformation vector contained two different genes (aphA6 and nptII) coding for two different enzymes. The aphA6 gene was regulated by the 16S rRNA promoter and gene 10 UTR capable of expression in the dark and in nongreen tissues. The nptII gene was regulated by the psbA promoter and UTR capable of expression in the light. Both genes with different regulatory sequences facilitated detoxification of the same selection agent (kanamycin) during day and night as well as in developing plastids and mature chloroplasts. The double barrel transformation vector was reported to be at least 8-fold more efficient than single gene (aphA6)-based chloroplast vectors.
To avoid potential disadvantages of antibiotic resistance genes, several studies have explored strategies for engineering chloroplasts that are free of antibiotic-resistance markers. The spinach (Spinacia oleracea) betaine aldehyde dehydrogenase (badh) gene has been developed as a plant-derived selectable marker gene to transform chloroplast genomes (Daniell et al., 2001b
The bacterial bar gene, encoding phosphinothricin acetyltransferase (PAT) and conferring herbicide resistance, has also been tested as a plastid-selectable marker. PAT served as an excellent marker in nuclear transformants and conferred resistance to the herbicide phosphinothricin. Expression of the bar gene in plastid conferred phosphinothricin resistance only when introduced by selection for a linked aadA gene. However, the bar gene was not found to be suitable for the direct selection of transplastomic lines, even when expressed at a higher level (approximately 7% of total soluble cellular protein). Thus, it shows that direct selection by herbicide resistance is constrained by way of subcellular localization of the gene encoding the detoxifying enzyme PAT (Lutz et al., 2001
A negative selection scheme has also been employed for plastid transformation based on expression of the bacterial gene codA (Serino and Maliga, 1997
GUS, chloramphenicol acetyl transferase, and GFP have been used as plastid reporters (Daniell and McFadden, 1987 ; Daniell et al., 1990; Ye et al., 1990; Khan and Maliga, 1999). The enzymatic activity of GUS can be visualized by histochemical staining (Ye et al., 1990; Daniell et al., 1991), whereas GFP is a visual marker that allows direct imaging of the fluorescent gene product in living cells. The GFP chromophore forms autocatalytically in the presence of oxygen and fluoresces green when absorbing blue or UV light (Hanson and Kohler, 2001). GFP has been used to detect transient gene expression (Hibberd et al., 1998) and stable transformation events (Reed et al., 2001; Lelivelt et al., 2005; Limaye et al., 2006) in chloroplasts. GFP has also been fused with AadA and used as a bifunctional visual and selectable marker (Khan and Maliga, 1999). Further, GFP has been used to test the concept of receptor-mediated oral delivery of foreign proteins. Cholera toxin B-subunit (CTB)-GFP fusion protein with a furin cleavage site in between CTB and GFP has been used to elucidate the path of CTB and GFP in the circulatory system (Limaye et al., 2006). Mice were fed with CTB-GFP-expressing plant leaf material. GFP was detected in the intestinal mucosa and submucosa, the hepatocytes of the liver, as well as various cells of spleen utilizing fluorescence microscopy and anti-GFP antibodies. In mice fed with untransformed leaf material or IFN-GFP fusion protein-expressing plant leaf material, no GFP fluorescence was observed. This confirmed the receptor-mediated oral delivery of a foreign protein (GFP) across the intestinal lumen into the systemic circulation. Moreover, GFP was not detected in any substantial amount in the liver or spleen of mice fed with IFN-GFP-expressing plants, suggesting that a transmucosal carrier such as CTB is required for delivery of an adequate amount of a foreign protein across the intestinal lumen into the systemic circulation. Thus, GFP has been used as a reporter gene in chloroplast expression and in animal studies.
Most of the studies involving plastid transformation have utilized antibiotic resistance gene for the recovery of transformed plastomes, but introducing such crops into the food chain may be a cause of concern. Strategies have been developed to eliminate antibiotic resistance genes after transformation, including homology-based excision via directly repeated sequences, excision by phage site-specific recombinases, transient co-integration of the marker gene, and cotransformation-segregation.
Early experiments with Chlamydomonas reinhardtii showed that homologous recombination between two direct repeats enabled marker removal under nonselective growth conditions (Fischer et al., 1996
So far, two recombinases (Cre and
The cotransformation-segregation approach involves transformation with two plasmids that target insertions at two different ptDNA locations: one plasmid carries a selective marker and the other a nonselected gene. Selection for the marker yields transplastomic clones that also bear an insertion of the nonselected gene. The prospect of the approach was first shown in C. reinhardtii (Kindle et al., 1991
Newly synthesized proteins are highly susceptible to proteases and require protection from chloroplast proteases. One such approach used the CRY chaperone (encoded by the orf2 gene) to fold the insecticidal protein, Cry2Aa2, into cuboidal crystals. The crystal structure protected the foreign proteins from degradation, thereby increasing protein accumulation over 128-fold (from 0.36% to 46.1% of total soluble protein [tsp]; De Cosa et al., 2001 ). Similarly, when the human serum albumin (hsa) coding sequence was regulated by the chloroplast psbA 5' and 3' UTRs in the light, protein expression increased 500-fold, resulting in the formation of protective inclusion bodies. A 3- to 10-fold reduction in HSA protein expression was seen when leaves were harvested in the dark (Fernandez-San Millan et al., 2003). This illustrated the power of regulatory sequences during illumination and protection from proteases when their access is limited.
Several studies on transgenic chloroplasts did not correlate increased transcript abundance with translation efficiency. For example, chloroplast-derived RbcS transcripts were measured to be 165-fold and 143-fold more than the nuclear RbcS antisense control plants when the transgene was regulated by the psbA 5' UTR or the promoterless gene 10 UTR, respectively. Although the psbA 5' UTR transgenic lines resulted in the first successful functional Rubisco in transgenic plants, the gene 10 UTR transgenic lines performed poorly (Dhingra et al., 2004
Human insulin was unstable in transgenic chloroplasts; fusion with CTB resulted in high-level expression (up to 16% tsp) and facilitated oral delivery studies to achieve protection against the development of insulitis in nonobese diabetic mice (Ruhlman et al., 2007
Tobacco has been the most widely exploited plastid transformation system because of its ease in genetic manipulations. A single tobacco plant is capable of generating a million seeds and 1 acre of tobacco can produce more than 40 metric tons of leaves per year (Cramer et al., 1999 ; Arlen et al., 2007). Harvesting leaves before flowering can offer nearly complete transgene containment in addition to protection offered by maternal inheritance. Recent studies have reported that escape of transgenes in tobacco is 0.0087% to 0.00024% (Daniell, 2007; Ruf et al., 2007; Svab and Maliga, 2007), making this an ideal system for use of chloroplasts as bioreactors. In addition, CMS has been engineered via the tobacco chloroplast genome as a failsafe method (Ruiz and Daniell, 2005). As a bioreactor, tobacco has been estimated to be more than 50 times less expensive than the frequently used Escherichia coli fermentation systems (Kusnadi et al., 1997). Additionally, tobacco eliminates contamination of food because it is a non-food and non-feed crop. Plastid transformation in higher plants was first successfully carried out in tobacco and is now a routine procedure because many foreign genes have been expressed to engineer agronomic traits, biopharmaceuticals, vaccines, or biomaterials (Table II
). However, presence of nicotine or other alkaloids has been a disadvantage for pharmaceutical production, but the chloroplast genome of low-nicotine varieties like LAMD has been used to engineer therapeutic proteins (Arlen et al., 2007). For oral delivery studies, there is a need to move beyond tobacco.
Extension of the plastid transformation technology to other species is important to exploit this platform. The study of DNA delivery strategies, target tissues, selection conditions, and regeneration systems is crucial for extending the range of species in which plastid transformation could be achieved. Plastid transformation is most commonly achieved by biolistic delivery of DNA into leaf explants but has also been achieved via direct DNA uptake by protoplasts (Lelivelt et al., 2005 ; Nugent et al., 2006). In species other than tobacco, like petunia and oilseed rape, adventitious shoot regeneration from bombarded leaf or petiole explants generated plastid transformants. Homoplasmic plants of soybean, carrot, and cotton were regenerated via somatic embryogenesis after bombardment of embryogenic calli, combined with the use of species-specific plastid vectors. Table I summarizes the chloroplast transformation method and selection conditions for different crop species. Attempts have been made in other plants (Table I) where protein production was carried out in non-green tissues such as micro-tuber (potato), fruit (tomato), and root (carrot). However, the amount of protein was lower than the level observed in leaf chloroplasts (Kumar et al., 2004a). Some progress has also been made in improving the chloroplast transformation system for tomato plants. Utilizing that plastid expression of a bacterial lycopene β-cyclase gene resulted in herbicide resistance and triggered conversion of lycopene, the main storage carotenoid of tomatoes, to β-carotene, resulting in a 4-fold enhancement of pro-vitamin A content of fruits (Wurbs et al., 2007). Stable chloroplast transformation system has also been reported for cabbage (Liu et al., 2007).
Recently, edible leafy crops, including lettuce, have attracted attention toward plastid genetic engineering. Edible plant species not only minimize downstream protein processing costs but also offer an ideal system for oral delivery. The leaves of lettuce are consumed raw by humans and the time from sowing seed to edible biomass is only weeks compared to months for crops such as tomato, potato, and carrot. Furthermore, lettuce is well suited for indoor cultivation by hydroculture systems (Kanamoto et al., 2006
Economically important crops such as carrot, cotton, and soybean regenerate in vitro through somatic embryogenesis (Daniell et al., 2005b
Plastid gene expression is regulated both at the transcriptional and posttranscriptional levels. Protein levels in chloroplasts depend on mRNA abundance, which is determined by promoter strength and mRNA stability. However, high mRNA levels do not result in high-level protein accumulation as posttranscriptional processes ultimately determine obtainable protein levels. Therefore, we have designed expression cassettes for transgene assembly to achieve optimal levels of protein accumulation in leaves (Fig. 1 ). The basic plastid transformation vector is comprised of flanking sequences and chloroplast-specific expression cassettes (Fig. 1). Species-specific chloroplast flanking sequence (e.g. trnI/trnA) is obtained by PCR using the primers designed from the available chloroplast genomes. The chloroplast expression cassette is composed of a promoter, selectable marker, and 5'/3' regulatory sequences to enhance the efficiency of transcription and translation of the gene. The chloroplast-specific promoters and regulatory elements are amplified from the total cellular DNA using primers designed on the basis of the sequence information available for the chloroplast genome. Suitable restriction sites are introduced to facilitate gene assembly.
Because of the high similarity in the transcription and translation systems between E. coli and chloroplasts, the chloroplast expression vectors are tested in E. coli first before proceeding with plant transformation. The growth of E. coli harboring the plastid transformation vector with the aadA gene in the presence of spectinomycin confirms expression of the aadA gene. Western blot with extracts from E. coli confirms expression of the gene of interest. Once expression of transgenes is confirmed in E. coli, the transformation vector is delivered into leaves (tobacco/lettuce) via particle bombardment. The leaves used for bombardment should be young, green, and healthy. The bombarded leaves are placed on selection medium with an appropriate concentration of antibiotics (RMOP in tobacco). Normally, in 3 to 10 weeks, putative transgenic shoots appear (Fig. 2, A and D). PCR analysis is used to screen the transgenic shoots and distinguish true chloroplast transgenic events from mutants or nuclear transgenic plants. Site-specific chloroplast integration of the transgene cassette is determined by using a set of primers, one of which anneals to the native chloroplast genome and the other anneals within the transgene cassette. Mutants and nuclear transgenic plants are not expected to produce a PCR product with these primers (Fig. 3A ). The leaf pieces from PCR-positive shoots are further selected for a second round to achieve homoplasmy (Fig. 2, B and E). The regenerated shoots are rooted with the same level of selection (Fig. 2, C and F) and checked for homoplasmy by Southern-blot analysis (Fig. 3B). The Southern blot is probed with radiolabeled flanking sequences used for homologous recombination. Transplastomic genome contains a larger size hybridizing fragment than the untransformed genome because of the presence of transgenes. If the transgenic plants are heteroplasmic, a native fragment is visible along with the larger transgenic fragment. Absence of the native fragment confirms the establishment of homoplasmy. Transgene expression is confirmed by western-blot analysis, and the effectiveness or properties or functionality of the introduced transgene is assessed. Seeds from the transgenic plants and untransformed plants are grown on spectinomycin-containing medium to check for maternal inheritance. Transgenic seeds germinate and grow into uniformly green plants. The absence of Mendelian segregation of transgenes confirms that they are maternally inherited to progeny.
Several useful transgenes have conferred valuable agronomic traits, including insect and pathogen resistance, drought tolerance, phytoremediation, salt tolerance, and CMS through chloroplast genetic engineering (Table II). Genetically engineered tobacco plants expressing an insecticidal protein Cry2Aa2 have shown resistance against target insects and insects that developed resistance against insecticidal protein (Kota et al., 1999 ). Expression of the Cry2Aa2 resulted in the utmost expression levels on record (approximately 46.1% of total leaf protein) and resulted in the detection of cuboidal crystals using transmission electron microscopy (De Cosa et al., 2001). In addition, soybean plastid transformants expressing Cry1Ab also conferred insecticidal activity against velvetbean caterpillar (Dufourmantel et al., 2005). The antimicrobial peptide MSI-99, an analog of magainin 2, was expressed via the chloroplast genome to obtain high levels of expression in transgenic tobacco plants. In planta assays with the bacterial pathogen Pseudomonas syringae pv tabaci and the fungal pathogen Colletotrichum destructivum showed necrotic lesions in untransformed control leaves, whereas transformed leaves showed no lesions (DeGray et al., 2001).
Environmental stress factors such as drought, salinity, and freezing are perilous to plants generally because of their sessile means of existence. Attempts to confer resistance to drought by expressing trehalose phosphate synthase 1 (tps1) gene via nuclear transformation have proven futile because of undesirable pleiotropic effects even at very low levels of trehalose accumulation. However, hyperexpression of tps1 in the chloroplasts has no phenotypic variation from the untransformed control plants, and transgenic seeds sprouted, grew, and remained green and healthy in drought tolerance bioassays with 3% to 6% PEG and dehydration/rehydration assays (Lee et al., 2003
Naturally occurring CMS has been documented for over 100 years for oilseed rape, maize (Zea mays), and rice. However, such systems are not available for the majority of crops used in agriculture. In presently available CMS lines, various loci in the nuclear genome direct a range of restoration factors that are not fully understood. Moreover, risk of sterility trait dilution through segregation and the production of transgenic seeds that spread transgenic traits to nontransgenic plants cannot be ruled out because of the possibility of cross-pollination of the male-sterile line with a restorer line or wild relative. To address some of these concerns, CMS has been engineered via introduction of phaA gene coding for β-ketothiolase into chloroplast genome. The transgenic lines were normal except for the male sterility phenotype lacking pollen (Ruiz and Daniell, 2005
Several chloroplast-derived biopharmaceutical proteins have been reported (Daniell, 2006 ; Table II). Stable expression of a pharmaceutical protein in chloroplasts was first reported for GVGVP, a protein-based polymer with medical uses such as wound coverings, artificial pericardia, and programmed drug delivery (Guda et al., 2000). Human ST (hST), a secretory protein, was expressed inside chloroplasts in a soluble, biologically active and disulfide-bonded form (Staub et al., 2000). The key use of hST is in the cure of hypopituitary dwarfism in children; additional indications are treatment of Turner syndrome, chronic renal failure, and human immunodeficiency virus wasting syndrome. Another important therapeutic protein that comprises approximately 60% of the protein in blood serum is HSA, prescribed in multigram quantities to restore blood volume in trauma and other clinical conditions. Early attempts at expressing HSA have achieved inadequately low levels of HSA (0.2% of tsp) in nuclear transgenic plants (Farran et al., 2002). On the other hand, in chloroplast transgenic plants, expression levels of up to 11.2% were observed (Fernandez-San Millan et al., 2003).
The type I IFNs are part of the body's first line of defense against viral attack and also invasion by bacterial pathogens, parasites, tumor cells, and allogeneic cells from grafts. IFN
As opposed to injected subunit vaccines, oral delivery and low-cost purification make plastid-derived subunit production quite plausible (Kamarajugadda and Daniell, 2006 ). Subunit vaccines expressed in plants are capable of inducing a mucosal response in animal models when given orally or parenterally; these animals also withstand a pathogen challenge. The ability for plant-derived vaccines to survive in the stomach is a major concern. However, bioencapsulation can protect the vaccine in the stomach and gradually releases the antigen in the gut (Mor et al., 1998). Vaccine antigens against cholera (Daniell et al., 2001a), tetanus (Tregoning et al., 2003), anthrax (Watson et al., 2004; Koya et al., 2005), plague (Daniell et al., 2005a), amebiasis (Chebolu and Daniell, 2007), and CPV (Molina et al., 2004) have been expressed in transgenic chloroplasts (Table II). For cholera, the CTB has been shown to be an extremely powerful vaccine candidate and is encoded by Vibrio cholerae. The chloroplast-expressed CTB assembled into pentameric protein and assumed correct quaternary structure for full activity. Subsequent binding assays confirmed the ability of chloroplast-derived CTB to bind to the intestinal membrane GM1 ganglioside receptors. CTB also acts as a powerful transmucosal carrier and is very effective in delivering several vaccine antigens. In one such investigation, oral administration of chloroplast-derived CTB-Pins fusion protein protected nonobese diabetic mice against development of insulitis (Ruhlman et al., 2007).
Recently, there has been an increased threat of bioterrorism in the post 9/11 world. Anthrax is always fatal if not treated immediately. Weapon-grade spores can be produced and stored for decades and can be spread by missiles, bombs, or even through the mail. Because of this, it is an ideal biological warfare agent. The currently available human vaccine for anthrax, derived from the culture supernatant of Bacillus anthracis, contains the protective antigen (PA) and traces of the lethal and edema factors. These factors may contribute to undesirable side effects linked with this vaccine. Therefore, an effective expression system that can provide a clean, safe, and efficacious vaccine is required. In an attempt to produce anthrax vaccine in large quantities and free of extraneous bacterial contaminants, PA was expressed in transgenic tobacco chloroplasts by inserting the pagA gene into the chloroplast genome (Watson et al., 2004
Besides vaccine antigens, biomaterial and amino acids have also been expressed in chloroplasts (Table II). Normally, p-hydroxybenzoic acid (pHBA) is produced in small quantities in all plants. In E. coli, the ubiC gene encoding chorismate pyruvate lyase catalyzes the direct conversion of chorismate to pyruvate and pHBA. However, in chloroplasts, chorismate is converted to pHBA by 10 consecutive enzymatic reactions due to lack of chorismate pyruvate lyase. Stable integration of the ubiC gene into the tobacco chloroplast resulted in hyperexpression of the enzyme and accumulation of this polymer up to 25% of dry weight (Viitanen et al., 2004 ). In another study, the gene for thermostable xylanase was expressed in the chloroplasts of tobacco plants (Leelavathi et al., 2003). Xylanase accumulated in the cells to approximately 6% of tsp. Zymography assay demonstrated that the estimated activity was 140,755 units kg–1 fresh leaf tissue.
Although the concept is more than 10 years old, plastid transformation has been accomplished in relatively few species. There are numerous factors that have hampered the expansion of chloroplast transformation technology to different plant species. One factor is the unavailability of the genome sequence. The chloroplast transformation vectors utilize homologous flanking regions for recombination and insertion of foreign genes. Therefore, there is an urgent need to sequence chloroplast genomes to facilitate transformation of crop species. Regardless of the small size of the genome and availability of tools to sequence an entire genome within a single day, it is hard to understand why only a few crop chloroplast genomes have been sequenced so far. Between 1986 and 2004, only six crop chloroplast genomes were sequenced. In the past 3 years, 25 new crop chloroplast genomes have been sequenced, including major crops like soybean and cotton (Saski et al., 2005 ; Lee et al., 2006a). Recent studies reveal that intergenic spacer regions and regulatory sequences contribute about 40% to 45% of the chloroplast genome and that spacer regions are not highly conserved. Comparison of nine grass chloroplast genomes revealed that not even one spacer region had 100% homology. Therefore, species-specific chloroplast vectors should be made for efficient transformation of grasses (Saski et al., 2007). Plastid transformation is a tissue culture-dependent process. Therefore, it is not adequate just to have the genome information; a better understanding of DNA delivery, selection, regeneration, and progression toward homoplasmy is essential to achieve plastid transformation in different taxonomic groups. Although chloroplast genome sequences of several monocots, including wheat and maize, have been available for several years, none of their genomes has been fully transformed so far. Major obstacles include the difficulty of expressing transgenes in non-green plastids, in which gene expression and gene regulation systems are quite distinct from those of mature green chloroplasts. Moreover, it is not possible to generate homoplasmic plants via subsequent rounds of regeneration using leaves as explants. Furthermore, proplastids are used as the transformation target rather than chloroplasts that are about 5-fold smaller in size than the fully developed chloroplasts in the green leaf tissues. Therefore, plastids with irreversible physical damage due to biolistic bombardment might be greater. It may also be necessary to develop new selection markers for a monocot-specific selection scheme. However, transformation of cotton or carrot using non-green embryogenic cells containing proplastids and regeneration via somatic embryogenesis offers new hopes for success.
We thank Dr. Nameirakpam Dolendro Singh and Tracey Ruhlman for assistance with figures. Received August 3, 2007; accepted September 30, 2007; published December 6, 2007.
1 This work was supported by the U.S. Department of Agriculture (grant no. 3611–21000–017–00D) and by the National Institutes of Health (grant no. 5R01 GM 63879–06).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Henry Daniell (daniell@mail.ucf.edu). www.plantphysiol.org/cgi/doi/10.1104/pp.107.106690 * Corresponding author; e-mail daniell{at}mail.ucf.edu.
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ADVANTAGES OF PLASTID...
GENOME ORGANIZATION AND CONCEPTS...
C31 phage integrase [Int]) have been tested for plastid marker gene excision. Using the P1 bacteriophage Cre/lox site-specific recombination system, a marker gene flanked by lox sites was removed after expression of the CRE protein was induced via the nuclear genome. The second site-specific recombinase, Int, appeared to be a better choice for the aadA marker gene removal when flanked with directly oriented nonidentical phage attP (215 bp) and bacterial attB (54 bp) attachment sites, which are recognized by Int recombinase. Efficient excision of the marker gene was shown after transformation of the nucleus with an int gene encoding plastid-targeted Int (Kittiwongwattana et al., 2007
(Leelavathi and Reddy, 2003
