Production of biologically active human basic fibroblast growth factor (hFGFb) using Nicotiana tabacum transplastomic plants.

MAIN CONCLUSION: We generated transplastomic tobacco lines that stably express a human Basic Fibroblast Growth Factor (hFGFb) in their chloroplasts stroma and purified a biologically active recombinant hFGFb. MAIN: The use of plants as biofactories presents as an attractive technology with the potential to efficiently produce high-value human recombinant proteins in a cost-effective manner. Plastid genome transformation stands out for its possibility to accumulate recombinant proteins at elevated levels. Of particular interest are recombinant growth factors, given their applications in animal cell culture and regenerative medicine. In this study, we produced recombinant human Fibroblast Growth Factor (rhFGFb), a crucial protein required for animal cell culture, in tobacco chloroplasts. We successfully generated two independent transplastomic lines that are homoplasmic and accumulate rhFGFb in their leaves. Furthermore, the produced rhFGFb demonstrated its biological activity by inducing proliferation in HEK293T cell lines. These results collectively underscore plastid genome transformation as a promising plant-based bioreactor for rhFGFb production.

Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants.

Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that chloroplast expression of many proteins, such as human epidermal growth factor (hEGF), results hindered by post-transcriptional mechanisms. hEGF degradation has been related to the redox potential of the stroma and protein misfolding. To solve this problem, we proposed the redirection of hEGF into the thylakoid lumen where the environment could improve disulfide bonds formation stabilizing the functional conformation of the protein. We generated transplastomic tobacco plants targeting hEGF protein to the thylakoid lumen by adding a transit peptide (Str). Following this approach, we could detect thylakoid lumen-targeted hEGF by western blotting while stromal accumulation of hEGF remained undetectable. Southern blot analysis confirmed the integration of the transgene through homologous recombination into the plastome. Northern blot analysis showed similar levels of egf transcripts in the EGF and StrEGF lines. These results suggest that higher stability of the hEGF peptide in the thylakoid lumen is the primary cause of the increased accumulation of the recombinant protein observed in StrEGF lines. They also highlight the necessity of exploring different sub-organellar destinations to improve the accumulation levels of a specific recombinant protein in plastids.

Expression of pathogenesis-related proteins in transplastomic tobacco plants confers resistance to filamentous pathogens under field trials.

Plants are continuously challenged by pathogens, affecting most staple crops compromising food security. They have evolved different mechanisms to counterattack pathogen infection, including the accumulation of pathogenesis-related (PR) proteins. These proteins have been implicated in active defense, and their overexpression has led to enhanced resistance in nuclear transgenic plants, although in many cases constitutive expression resulted in lesion-mimic phenotypes. We decided to evaluate plastid transformation as an alternative to overcome limitations observed for nuclear transgenic technologies. The advantages include the possibilities to express polycistronic RNAs, to obtain higher protein expression levels, and the impeded gene flow due to the maternal inheritance of the plastome. We transformed Nicotiana tabacum plastids to co-express the tobacco PR proteins AP24 and ß-1,3-glucanase. Transplastomic tobacco lines were characterized and subsequently challenged with Rhizoctonia solani, Peronospora hyoscyami f.sp. tabacina and Phytophthora nicotianae. Results showed that transplastomic plants expressing AP24 and ß-1,3-glucanase are resistant to R. solani in greenhouse conditions and, furthermore, they are protected against P.hyoscyami f.sp. tabacina and P. nicotianae in field conditions under high inoculum pressure. Our results suggest that plastid co- expression of PR proteins AP24 and ß-1,3-glucanase resulted in enhanced resistance against filamentous pathogens.

Selective pressure against horizontally acquired prokaryotic genes as a driving force of plastid evolution.

The plastid organelle comprises a high proportion of nucleus-encoded proteins that were acquired from different prokaryotic donors via independent horizontal gene transfers following its primary endosymbiotic origin. What forces drove the targeting of these alien proteins to the plastid remains an unresolved evolutionary question. To better understand this process we screened for suitable candidate proteins to recapitulate their prokaryote-to-eukaryote transition. Here we identify the ancient horizontal transfer of a bacterial polyphenol oxidase (PPO) gene to the nuclear genome of an early land plant ancestor and infer the possible mechanism behind the plastidial localization of the encoded enzyme. Arabidopsis plants expressing PPO versions either lacking or harbouring a plastid-targeting signal allowed examining fitness consequences associated with its subcellular localization. Markedly, a deleterious effect on plant growth was highly correlated with PPO activity only when producing the non-targeted enzyme, suggesting that selection favoured the fixation of plastid-targeted protein versions. Our results reveal a possible evolutionary mechanism of how selection against heterologous genes encoding cytosolic proteins contributed in incrementing plastid proteome complexity from non-endosymbiotic gene sources, a process that may also impact mitochondrial evolution.

Expression of the Multimeric and Highly Immunogenic Brucella spp. Lumazine Synthase Fused to Bovine Rotavirus VP8d as a Scaffold for Antigen Production in Tobacco Chloroplasts.

Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein which can accommodate foreign polypeptides or protein domains fused to its N-termini, markedly increasing their immunogenicity. The inner core domain (VP8d) of VP8 spike protein from bovine rotavirus is responsible for viral adhesion to sialic acid residues and infection. It also displays neutralizing epitopes, making it a good candidate for vaccination. In this work, the BLS scaffold was assessed for the first time in plants for recombinant vaccine development by N-terminally fusing BLS to VP8d and expressing the resulting fusion (BLSVP8d) in tobacco chloroplasts. Transplastomic plants were obtained and characterized by Southern, northern and western blot. BLSVP8d was highly expressed, representing 40% of total soluble protein (4.85 mg/g fresh tissue). BLSVP8d remained soluble and stable during all stages of plant development and even in lyophilized leaves stored at room temperature. Soluble protein extracts from fresh and lyophilized leaves were able to induce specific neutralizing IgY antibodies in a laying hen model. This work presents BLS as an interesting platform for highly immunogenic injectable, or even oral, subunit vaccines. Lyophilization of transplastomic leaves expressing stable antigenic fusions to BLS would further reduce costs and simplify downstream processing, purification and storage, allowing for more practical vaccines.

Translational fusion and redirection to thylakoid lumen as strategies to enhance accumulation of human papillomavirus E7 antigen in tobacco chloroplasts.

Human papillomavirus (HPV) is the causal agent of cervical cancer, one of the most common causes of death in women worldwide, and its E7 antigen is the major candidate for a therapeutic vaccine. The large scale production of E7 by molecular farming that would lead to the development of a safe and inexpensive vaccine is impaired by its low accumulation level in the plant cell. To enhance antigen production in the plastids, two alternative strategies were carried out: the expression of E7 as a translational fusion to ß-glucuronidase enzyme and redirection of E7 into the thylakoid lumen. The use of the ß-glucuronidase as a partner protein turned out to be a successful strategy, antigen expression levels were enhanced between 30 and 40 times relative to unfused E7. Moreover, best accumulation, albeit at a high metabolic cost that compromised biomass production, was obtained redirecting E7 into the thylakoid lumen by the incorporation of the N-terminal transit peptide, Str. Following this approach lumenal E7 production exceeded the stromal by two orders of magnitude. Our results highlight the relevance of exploring different strategies to improve recombinant protein stability for certain transgenes in order to exploit potential advantages of recombinant protein accumulation in chloroplasts.

Expression of the minor isoform pea ferredoxin in tobacco alters photosynthetic electron partitioning and enhances cyclic electron flow.

Ferredoxins (Fds) are ferrosulfoproteins that function as low-potential electron carriers in plants. The Fd family is composed of several isoforms that share high sequence homology but differ in functional characteristics. In leaves, at least two isoforms conduct linear and cyclic photosynthetic electron transport around photosystem I, and mounting evidence suggests the existence of at least partial division of duties between these isoforms. To evaluate the contribution of different kinds of Fds to the control of electron fluxes along the photosynthetic electron transport chain, we overexpressed a minor pea (Pisum sativum) Fd isoform (PsFd1) in tobacco (Nicotiana tabacum) plants. The transplastomic OeFd1 plants exhibited variegated leaves and retarded growth and developmental rates. Photosynthetic studies of these plants indicated a reduction in carbon dioxide assimilation rates, photosystem II photochemistry, and linear electron flow. However, the plants showed an increase in nonphotochemical quenching, better control of excitation pressure at photosystem II, and no evidence of photoinhibition, implying a better dynamic regulation to remove excess energy from the photosynthetic electron transport chain. Finally, analysis of P700 redox status during illumination confirmed that the minor pea Fd isoform promotes enhanced cyclic flow around photosystem I. The two novel features of this work are: (1) that Fd levels achieved in transplastomic plants promote an alternative electron partitioning even under greenhouse light growth conditions, a situation that is exacerbated at higher light intensity measurements; and (2) that an alternative, minor Fd isoform has been overexpressed in plants, giving new evidence of labor division among Fd isoforms.

Flavodoxin displays dose-dependent effects on photosynthesis and stress tolerance when expressed in transgenic tobacco plants.

Ferredoxins are iron-sulfur proteins involved in various one-electron transfer pathways. Ferredoxin levels decrease under adverse environmental conditions in photosynthetic organisms. In cyanobacteria, this decline is compensated by induction of flavodoxin, an isofunctional flavoprotein. Flavodoxin is not present in higher plants, but transgenic Nicotiana tabacum lines accumulating Anabaena flavodoxin in plastids display increased tolerance to different sources of environmental stress. As the degree of tolerance correlated with flavodoxin dosage in plastids of nuclear-transformed transgenic tobacco, we prepared plants expressing even higher levels of flavodoxin by direct plastid transformation. A suite of nuclear- and chloroplast-transformed lines expressing a wide range of flavodoxin levels, from 0.3 to 10.8 µmol m(-2), did not exhibit any detectable growth phenotype relative to the wild type. In the absence of stress, the contents of both chlorophyll a and carotenoids, as well as the photosynthetic performance (photosystem II maximum efficiency, photosystem II operating efficiency, electron transport rates and carbon assimilation rates), displayed a moderate increase with flavodoxin concentrations up to 1.3-2.6 µmol flavodoxin m(-2), and then declined to wild-type levels. Stress tolerance, as estimated by the damage inflicted on exposure to the pro-oxidant methyl viologen, also exhibited a bell-shaped response, with a significant, dose-dependent increase in tolerance followed by a drop in the high-expressing lines. The results indicate that optimal photosynthetic performance and stress tolerance were observed at flavodoxin levels comparable to those of endogenous ferredoxin. Further increases in flavodoxin content become detrimental to plant fitness.

Translational fusion and redirection to thylakoid lumen as strategies to improve the accumulation of a camelid antibody fragment in transplastomic tobacco.

Fragments from camelid single-chain antibodies known as VHHs or nanobodies represent a valuable tool in diagnostics, investigation and passive immunity therapy. Here, we explored different strategies to improve the accumulation of a neutralizing VHH antibody against rotavirus in tobacco transplastomic plants. First, we attempted to express the VHH in the chloroplast stroma and then two alternative strategies were carried out to improve the expression levels: expression as a translational fusion to the ß-glucuronidase enzyme (GUS-E-VHH), and redirection of the VHH into the thylakoid lumen (pep-VHH). Every attempt to produce transplastomic plants expressing the VHH in the stroma was futile. The transgene turned out to be unstable and the presence of the VHH protein was almost undetectable. Although pep-VHH plants also presented some of the aforementioned problems, higher accumulation of the nanobody was observed (2-3% of the total soluble proteins). The use of ß-glucuronidase as a partner protein turned out to be a successful strategy and expression levels reached 3% of the total soluble proteins. The functionality of the VHHs produced by pep-VHH and GUS-E-VHH plants was studied and compared with that of the antibody produced in Escherichia coli. This work contributes to optimizing the expression of VHH in transplastomic plants. Recombinant proteins could be obtained either by accumulation in the thylakoid lumen or as a fusion protein with ß-glucuronidase, and both strategies allow for further optimization.

Stacking of antimicrobial genes in potato transgenic plants confers increased resistance to bacterial and fungal pathogens.

Solanum tuberosum plants were transformed with three genetic constructions expressing the Nicotiana tabacum AP24 osmotine, Phyllomedusa sauvagii dermaseptin and Gallus gallus lysozyme, and with a double-transgene construction expressing the AP24 and lysozyme sequences. Re-transformation of dermaseptin-transformed plants with the AP24/lysozyme construction allowed selection of plants simultaneously expressing the three transgenes. Potato lines expressing individual transgenes or double- and triple-transgene combinations were assayed for resistance to Erwinia carotovora using whole-plant and tuber infection assays. Resistance levels for both infection tests compared consistently for most potato lines and allowed selection of highly resistant phenotypes. Higher resistance levels were found in lines carrying the dermaseptin and lysozyme sequences, indicating that theses proteins are the major contributors to antibacterial activity. Similar results were obtained in tuber infection tests conducted with Streptomyces scabies. Plant lines showing the higher resistance to bacterial infections were challenged with Phytophthora infestans, Rhizoctonia solani and Fusarium solani. Considerable levels of resistance to each of these pathogens were evidenced employing semi-quantitative tests based in detached-leaf inoculation, fungal growth inhibition and in vitro plant inoculation. On the basis of these results, we propose that stacking of these transgenes is a promising approach to achieve resistance to both bacterial and fungal pathogens.

Transformation of Solanum tuberosum plastids allows high expression levels of ß-glucuronidase both in leaves and microtubers developed in vitro.

Plastid genome transformation offers an attractive methodology for transgene expression in plants, but for potato, only expression of gfp transgene (besides the selective gene aadA) has been published. We report here successful expression of ß-glucuronidase in transplastomic Solanum tuberosum (var. Desiree) plants, with accumulation levels for the recombinant protein of up to 41% of total soluble protein in mature leaves. To our knowledge, this is the highest expression level reported for a heterologous protein in S. tuberosum. Accumulation of the recombinant protein in soil-grown minitubers was very low, as described in previous reports. Interestingly, microtubers developed in vitro showed higher accumulation of ß-glucuronidase. As light exposure during their development could be the trigger for this high accumulation, we analyzed the effect of light on ß-glucuronidase accumulation in transplastomic tubers. Exposure to light for 8 days increased ß-glucuronidase accumulation in soil-grown tubers, acting as a light-inducible expression system for recombinant protein accumulation in tuber plastids. In this paper we show that plastid transformation in potato allows the highest recombinant protein accumulation in foliar tissue described so far for this food crop. We also demonstrate that in tubers high accumulation is possible and depends on light exposure. Because tubers have many advantages as protein storage organs, these results could lead to new recombinant protein production schemes based on potato.

Field testing, gene flow assessment and pre-commercial studies on transgenic Solanum tuberosum spp. tuberosum (cv. Spunta) selected for PVY resistance in Argentina.

Solanum tuberosum ssp. tuberosum (cv. Spunta) was transformed with a chimeric transgene containing the Potato virus Y (PVY) coat protein (CP) sequence. Screening for PVY resistance under greenhouse conditions yielded over 100 independent candidate lines. Successive field testing of selected lines allowed the identification of two genetically stable PVY-resistant lines, SY230 and SY233, which were further evaluated in field trials at different potato-producing regions in Argentina. In total, more than 2,000 individuals from each line were tested along a 6-year period. While no or negligible PVY infection was observed in the transgenic lines, infection rates of control plants were consistently high and reached levels of up to 70-80%. Parallel field studies were performed in virus-free environments to assess the agronomical performance of the selected lines. Tubers collected from these assays exhibited agronomical traits and biochemical compositions indistinguishable from those of the non-transformed Spunta cultivar. In addition, an interspecific out-crossing trial to determine the magnitude of possible natural gene flow between transgenic line SY233 and its wild relative Solanum chacoense was performed. This trial yielded negative results, suggesting an extremely low probability for such an event to occur.

Cyanobacterial flavodoxin complements ferredoxin deficiency in knocked-down transgenic tobacco plants.

Ferredoxins are the main electron shuttles in chloroplasts, accepting electrons from photosystem I and delivering them to essential oxido-reductive pathways in the stroma. Ferredoxin levels decrease under adverse environmental conditions in both plants and photosynthetic micro-organisms. In cyanobacteria and some algae, this decrease is compensated for by induction of flavodoxin, an isofunctional flavoprotein that can replace ferredoxin in many reactions. Flavodoxin is not present in plants, but tobacco lines expressing a plastid-targeted cyanobacterial flavodoxin developed increased tolerance to environmental stress. Chloroplast-located flavodoxin interacts productively with endogenous ferredoxin-dependent pathways, suggesting that its protective role results from replacement of stress-labile ferredoxin. We tested this hypothesis by using RNA antisense and interference techniques to decrease ferredoxin levels in transgenic tobacco. Ferredoxin-deficient lines showed growth arrest, leaf chlorosis and decreased CO(2) assimilation. Chlorophyll fluorescence measurements indicated impaired photochemistry, over-reduction of the photosynthetic electron transport chain and enhanced non-photochemical quenching. Expression of flavodoxin from the nuclear or plastid genome restored growth, pigment contents and photosynthetic capacity, and relieved the electron pressure on the electron transport chain. Tolerance to oxidative stress also recovered. In the absence of flavodoxin, ferredoxin could not be decreased below 45% of physiological content without fatally compromising plant survival, but in its presence, lines with only 12% remaining ferredoxin could grow autotrophically, with almost wild-type phenotypes. The results indicate that the stress tolerance conferred by flavodoxin expression in plants stems largely from functional complementation of endogenous ferredoxin by the cyanobacterial flavoprotein.

Safety assessment of nonbrowning potatoes: opening the discussion about the relevance of substantial equivalence on next generation biotech crops.

It is expected that the next generation of biotech crops displaying enhanced quality traits with benefits to both farmers and consumers will have a better acceptance than first generation biotech crops and will improve public perception of genetic engineering. This will only be true if they are proven to be as safe as traditionally bred crops. In contrast with the first generation of biotech crops where only a single trait is modified, the next generation of biotech crops will add a new level of complexity inherent to the mechanisms underlying their output traits. In this study, a comprehensive evaluation of the comparative safety approach on a quality-improved biotech crop with metabolic modifications is presented. Three genetically engineered potato lines with silenced polyphenol oxidase (Ppo) transcripts and reduced tuber browning were characterized at both physiological and molecular levels and showed to be equivalent to wild-type (WT) plants when yield-associated traits and photosynthesis were evaluated. Analysis of the primary metabolism revealed several unintended metabolic modifications in the engineered tubers, providing evidence for potential compositional inequivalence between transgenic lines and WT controls. The silencing construct sequence was in silico analysed for potential allergenic cross-reactivity, and no similarities to known allergenic proteins were identified. Moreover, in vivo intake safety evaluation showed no adverse effects in physiological parameters. Taken together, these results provide the first evidence supporting that the safety of next generation biotech crops can be properly assessed following the current evaluation criterion, even if the transgenic and WT crops are not substantially equivalent.

Improvement of aroma in transgenic potato as a consequence of impairing tuber browning.

Sensory analysis studies are critical in the development of quality enhanced crops, and may be an important component in the public acceptance of genetically modified foods. It has recently been established that odor preferences are shared between humans and mice, suggesting that odor exploration behavior in mice may be used to predict the effect of odors in humans. We have previously found that mice fed diets supplemented with engineered nonbrowning potatoes (-PPO) consumed more potato than mice fed diets supplemented with wild-type potatoes (WT). This prompted us to explore a possible role of potato odor in mice preference for nonbrowning potatoes. Taking advantage of two well established neuroscience paradigms, the "open field test" and the "nose-poking preference test", we performed experiments where mice exploration behavior was monitored in preference assays on the basis of olfaction alone. No obvious preference was observed towards -PPO or WT lines when fresh potato samples were tested. However, when oxidized samples were tested, mice consistently investigated -PPO potatoes more times and for longer periods than WT potatoes. Congruently, humans discriminated WT from -PPO samples with a considerably better performance when oxidized samples were tested than when fresh samples were tested in blind olfactory experiments. Notably, even though participants ranked all samples with an intermediate level of pleasantness, there was a general consensus that the -PPO samples had a more intense odor and also evoked the sense-impression of a familiar vegetable more often than the WT samples. Taken together, these findings suggest that our previous observations might be influenced, at least in part, by differential odors that are accentuated among the lines once oxidative deterioration takes place. Additionally, our results suggest that nonbrowning potatoes, in addition to their extended shelf life, maintain their odor quality for longer periods of time than WT potatoes. To our knowledge this is the first report on the use of an animal model applied to the sensory analysis of a transgenic crop.

High expression level of a foot and mouth disease virus epitope in tobacco transplastomic plants.

Chloroplast transformation has an extraordinary potential for antigen production in plants because of the capacity to accumulate high levels of recombinant proteins and increased biosafety due to maternal plastid inheritance in most crops. In this article, we evaluate tobacco chloroplasts transformation for the production of a highly immunogenic epitope containing amino acid residues 135-160 of the structural protein VP1 of the foot and mouth disease virus (FMDV). To increase the accumulation levels, the peptide was expressed as a fusion protein with the beta-glucuronidase reporter gene (uidA). The recombinant protein represented the 51% of the total soluble proteins in mature leaves, a level higher than those of the Rubisco large subunit, the most abundant protein in the leaf of a wild-type plant. Despite this high accumulation of heterologous protein, the transplastomic plants and wild-type tobacco were phenotypically indistinguishable. The FMDV epitope expressed in transplastomic plants was immunogenic in mice. These results show that transplastomic tobacco express efficiently the recombinant protein, and we conclude that this technology allows the production of large quantities of immunogenic proteins.

Potato virus X coat protein fusion to human papillomavirus 16 E7 oncoprotein enhance antigen stability and accumulation in tobacco chloroplast.

Cervical cancer linked to infection with human papillomavirus (HPV) is the third cause of cancer-related death in women. As the virus cannot be propagated in culture, vaccines have been based on recombinant antigens with inherited high-cost production. In a search of alternative cheap production system, E7 HPV type 16 protein, an attractive candidate for anticancer vaccine development, was engineered to be expressed in tobacco chloroplast. In addition, E7 coding sequence was fused to potato virus X coat protein (CP) to compare expression level. Results show that E7CP transcript accumulation reached lower levels than non-fused E7. However, antigen expression levels were higher for fusion protein indicating that CP stabilizes E7 peptide in the chloroplast stroma. These results support viability of transplastomic plants for antigen production and the relevance of improving recombinant peptide stability for certain transgenes to enhance protein accumulation in this organelle.

Accumulation of hEGF and hEGF-fusion proteins in chloroplast-transformed tobacco plants is higher in the dark than in the light.

Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that heterologous protein accumulation in chloroplasts could be hindered by post-transcriptional mechanisms not yet characterized. Here, we describe the development and characterization of transplastomic tobacco plants transformed with four different transformation vectors for the expression of human epidermal growth factor (hEGF). We showed that, although the corresponding transcript was present in all of the analyzed plants, hEGF could only be detected when fused to the first 186 amino acids of bacterial beta-glucuronidase (GUS). In addition, we observed that the expression levels of recombinant protein increased when plants were placed in the dark or when leaves were incubated in the presence of electron transport inhibitors, such as methyl viologen (MV) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). These results suggest that the mechanism responsible for hEGF instability in chloroplasts is regulated by light.

Immunological Detection of a GarV-Type Virus in Argentine Garlic Cultivars.

A cDNA library representing the genomes of several garlic viruses was generated using a viral RNA mixture as template. Analysis of randomly selected clones allowed the identification of different viral genomic sequences. On the basis of amino acid and nucleotide sequence comparisons, one of them was assigned to garlic virus A (GarV-A), a novel flexuous, rod-shaped virus recently reported in Japan. The coat protein (CP) of this virus was expressed in Escherichia coli cells and used as immunogen to produce polyclonal antibodies. The expression protein was recognized by an antiserum detecting garlic mite-borne filamentous virus, but did not react with antibodies specific for other garlic viruses. Antibodies raised against the viral CP reacted with extracts infected with garlic mite-borne viruses and fail to recognize preparations of onion yellow dwarf potyvirus, leek yellow stripe potyvirus, and carnation latent carlavirus. The same antibodies decorated viral particles exhibiting a modal length of 586 nm in immuno electron microscopy with decoration assays. Double-antibody enzyme-linked immunosorbent assays and tissue printing assays performed with these antibodies allowed detection of GarV-A in most garlic cultivars used in Argentina.