In silico designing and characterization of outer membrane protein K (OmpK) from Vibrio anguillarum and its expression in Nicotiana tabacum for the development of a plant-based vaccine against fish vibriosis.

Vibriosis is caused by Vibrio anguillarum in various species of aquaculture. A novel, secure, and stable vaccine is needed to eradicate vibriosis. Here, for reverse vaccinology and plant-based expression, the outer membrane protein K (OmpK) of V. anguillarum was chosen due to its conserved nature in all Vibrio species. OmpK, an ideal vaccine candidate against vibriosis, demonstrated immunogenic, non-allergic, and non-toxic behavior by using various bioinformatics tools. Docking showed the interaction of the OmpK model with TLR-5. In comparison to costly platforms, plants can be used as alternative and economic bio-factories to produce vaccine antigens. We expressed OmpK antigen in Nicotiana tabacum using Agrobacterium-mediated transformation. The expression vector was constructed using Gateway® cloning. Transgene integration was verified by polymerase chain reaction (PCR), and the copy number via qRT-PCR, which showed two copies of transgenes. Western blotting detected monomeric form of OmpK protein. The total soluble protein (TSP) fraction of OmpK was equivalent to 0.38% as detected by ELISA. Mice and fish were immunized with plant-derived OmpK antigen, which showed a significantly high level of anti-OmpK antibodies. The present study is the first report of OmpK antigen expression in higher plants for the potential use as vaccine in aquaculture against vibriosis, which could provide protection against multiple Vibrio species due to the conserved nature OmpK antigen.

Inducible expression of human papillomavirus-16 L1 capsomeres in the plastomes of Nicotiana tabacum: Transplastomic plants develop normal flowers and pollen.

Human papillomavirus type-16 (HPV-16) is the major HPV type involved in causing cervical cancer among women. The disease burden is high in developing and underdeveloped countries. Previously, the constitutive expression of HPV-16 L1 protein led to male sterility in transplastomic tobacco plants. Here, the HPV-16 L1 gene was expressed in chloroplasts of Nicotiana tabacum under the control of an ethanol-inducible promoter, trans-activated by nucleus-derived signal peptide. Plants containing nuclear component were transformed with transformation vector pEXP-T7-L1 by biolistic gun. The transformation and homoplasmic status of transformed plants was verified by polymerase chain reaction and Southern blotting, respectively. Protein was induced by spraying 5% ethanol for 7 consecutive days. The correct folding of L1 protein was confirmed by antigen-capture ELISA using a conformation-specific antibody. The L1 protein accumulated up to 3 µg/g of fresh plant material. The L1 protein was further purified using affinity chromatography. All transplastomic plants developed normal flowers and produced viable seeds upon self-pollination. Pollens also showed completely normal structure under light microscope and scanning electron microscopy. These data confirm the use of the inducible expression as plant-safe approach for expressing transgenes in plants, especially those genes that cause detrimental effects on plant growth and morphology.

Plastidial Expression of 3ß-Hydroxysteroid Dehydrogenase and Progesterone 5ß-Reductase Genes Confer Enhanced Salt Tolerance in Tobacco.

The short-chain dehydrogenase/reductase (SDR) gene family is widely distributed in all kingdoms of life. The SDR genes, 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and progesterone 5-ß-reductases (P5ßR1, P5ßR2) play a crucial role in cardenolide biosynthesis pathway in the Digitalis species. However, their role in plant stress, especially in salinity stress management, remains unexplored. In the present study, transplastomic tobacco plants were developed by inserting the 3ß-HSD, P5ßR1 and P5ßR2 genes. The integration of transgenes in plastomes, copy number and transgene expression at transcript and protein level in transplastomic plants were confirmed by PCR, end-to-end PCR, qRT-PCR and Western blot analysis, respectively. Subcellular localization analysis showed that 3ß-HSD and P5ßR1 are cytoplasmic, and P5ßR2 is tonoplast-localized. Transplastomic lines showed enhanced growth in terms of biomass and chlorophyll content compared to wild type (WT) under 300 mM salt stress. Under salt stress, transplastomic lines remained greener without negative impact on shoot or root growth compared to the WT. The salt-tolerant transplastomic lines exhibited enhanced levels of a series of metabolites (sucrose, glutamate, glutamine and proline) under control and NaCl stress. Furthermore, a lower Na+/K+ ratio in transplastomic lines was also observed. The salt tolerance, mediated by plastidial expression of the 3ß-HSD, P5ßR1 and P5ßR2 genes, could be due to the involvement in the upregulation of nitrogen assimilation, osmolytes as well as lower Na+/K+ ratio. Taken together, the plastid-based expression of the SDR genes leading to enhanced salt tolerance, which opens a window for developing saline-tolerant plants via plastid genetic engineering.

Plastid genomics of Nicotiana (Solanaceae): insights into molecular evolution, positive selection and the origin of the maternal genome of Aztec tobacco (Nicotiana rustica).

Species of the genus Nicotiana (Solanaceae), commonly referred to as tobacco plants, are often cultivated as non-food crops and garden ornamentals. In addition to the worldwide production of tobacco leaves, they are also used as evolutionary model systems due to their complex development history tangled by polyploidy and hybridization. Here, we assembled the plastid genomes of five tobacco species: N. knightiana, N. rustica, N. paniculata, N. obtusifolia and N. glauca. De novo assembled tobacco plastid genomes had the typical quadripartite structure, consisting of a pair of inverted repeat (IR) regions (25,323-25,369 bp each) separated by a large single-copy (LSC) region (86,510-86,716 bp) and a small single-copy (SSC) region (18,441-18,555 bp). Comparative analyses of Nicotiana plastid genomes with currently available Solanaceae genome sequences showed similar GC and gene content, codon usage, simple sequence and oligonucleotide repeats, RNA editing sites, and substitutions. We identified 20 highly polymorphic regions, mostly belonging to intergenic spacer regions (IGS), which could be suitable for the development of robust and cost-effective markers for inferring the phylogeny of the genus Nicotiana and family Solanaceae. Our comparative plastid genome analysis revealed that the maternal parent of the tetraploid N. rustica was the common ancestor of N. paniculata and N. knightiana, and the later species is more closely related to N. rustica. Relaxed molecular clock analyses estimated the speciation event between N. rustica and N. knightiana appeared 0.56 Ma (HPD 0.65-0.46). Biogeographical analysis supported a south-to-north range expansion and diversification for N. rustica and related species, where N. undulata and N. paniculata evolved in North/Central Peru, while N. rustica developed in Southern Peru and separated from N. knightiana, which adapted to the Southern coastal climatic regimes. We further inspected selective pressure on protein-coding genes among tobacco species to determine if this adaptation process affected the evolution of plastid genes. These analyses indicate that four genes involved in different plastid functions, including DNA replication (rpoA) and photosynthesis (atpB, ndhD and ndhF), came under positive selective pressure as a result of specific environmental conditions. Genetic mutations in these genes might have contributed to better survival and superior adaptations during the evolutionary history of tobacco species.

Chloroplast-based inducible expression of ESAT-6 antigen for development of a plant-based vaccine against tuberculosis.

Mycobacterium tuberculosis causes tuberculosis in humans. The major disease burden of tuberculosis lies in developing countries. Lack of an effective vaccine for adults is one of the major hurdles for controlling this deadly disease. In the present study, 6 kDa early secretory antigenic target (ESAT-6) of M. tuberculosis was inducibly expressed in chloroplasts of Nicotiana tabacum. The expression of ESAT-6 in chloroplasts was controlled by T7 promoter that was activated by nuclear-generated signal peptide. Tobacco plants, containing nuclear component, were transformed via biolistic bombardment with pEXP-T7-ESAT-6 obtained by Gateway® cloning. Transformation and homoplasmic status of transplastomic plants was confirmed by polymerase chain reaction and Southern blotting. Plants were induced for protein expression by spraying with 5% ethanol for 1 day, 3 days, 7 days and 10 days. ESAT-6 protein was detected by immunoblot analysis and maximum protein was obtained for 10 days induced plants that was estimated to accumulate up to 1.2% of total soluble fraction of protein. Transplastomic plants showed completely normal morphology. Transplastomic and untransformed plants became slightly chlorotic upon prolonged exposure to ethanol until 10 days. Taken together, this data could help in the development of an antigen-based subunit vaccine against tuberculosis.

Effect of Rol Genes on Polyphenols Biosynthesis in Artemisia annua and Their Effect on Antioxidant and Cytotoxic Potential of the Plant.

Flavonoids are famous for their antioxidant capacity and redox potential. They can combat with cell aging, lipid peroxidation, and cancer. In the present study, Artemisia annua hybrid (Hyb8001r) was subjected to qualitative and quantitative analysis of flavonoids through HPLC. Rol genes transgenics of A. annua were also evaluated for an increase in their flavonoid content along with an increase in antioxidant and cytotoxic potential. This was also correlated with the expression level of flavonoids biosynthetic pathway genes as determined by real-time qPCR. Phenylalanine ammonia-lyase and chalcone synthase genes were found to be significantly more highly expressed in rol B (four to sixfold) and rol C transgenics (3.8-5.5-fold) than the wild-type plant. Flavonoids detected in the wild-type A. annua through HPLC include rutin (0.31 mg/g DW), quercetin (0.01 mg/g DW), isoquercetin (0.107 mg/g DW) and caffeic acid (0.03 mg/g DW). Transgenics of the rol B gene showed up to threefold increase in rutin and caffeic acid, sixfold increase in isoquercetin, and fourfold increase in quercetin. Whereas, in the case of transgenics of rol C gene, threefold increase in rutin and quercetin, 5 fold increase in isoquercetin, and 2.6-fold increase in caffeic acid was followed. Total phenolics and flavonoids content was also found to be increased in rol B (1.5-fold) and rol C (1.4-fold) transgenics as compared to the wild-type plant along with increased free radical scavenging activity. Similarly, the cytotoxic potential of rol gene transgenics against MCF7, HeLA, and HePG2 cancer cell lines was found to be significantly enhanced than the wild-type plant of A. annua. Current findings support the fact that rol genes can alter the secondary metabolism and phytochemical level of the plant. They increased the flavonoids content of A. annua by altering the expression level of flavonoids biosynthetic pathway genes. Increased flavonoid content also enhanced the antioxidant and cytotoxic potential of the plant.

Expression of HPV-16 L1 capsomeres with glutathione-S-transferase as a fusion protein in tobacco plastids: an approach for a capsomere-based HPV vaccine.

Human Papillomavirus (HPV) is the main cause of cervical cancer, which is the second most severe cancer of women worldwide, particularly in developing countries. Although vaccines against HPV infection are commercially available, they are neither affordable nor accessible to women in low income countries e.g. Africa. Thus, alternative cost-effective vaccine production approaches need to be developed. This study uses tobacco plants to express pentameric capsomeres of HPV that have been reported to generate elevated immune responses against HPV. A modified HPV-16 L1 (L1_2xCysM) protein has been expressed as a fusion protein with glutathione-S-transferase (GST) in tobacco chloroplasts following biolistic transformation. In total 7 transplastomic lines with healthy phenotypes were generated. Site specific integration of the GST-L1_2xCysM and aadA genes was confirmed by PCR. Southern blot analysis verified homogenous transformation of all transplastomic lines. Antigen capture ELISA with the conformation-specific antibody Ritti01, showed protein expression as well as the retention of immunogenic epitopes of L1 protein. In their morphology, GST-L1 expressing tobacco plants were identical to wild type plants and yielded fertile flowers. Taken together, these data enrich knowledge for future development of cost-effective plant-made vaccines against HPV.

A novel chloroplast transformation vector compatible with the Gateway(®) recombination cloning technology.

To analyze the suitability of Gateway(®) vectors for transformation of chloroplasts, we converted a standard plastid transformation vector into a Gateway(®) destination vector containing the necessary recombination sites attR1 and attR2. Insertion of the green fluorescent protein (GFP) coding sequence with associated T7g10 ribosome binding site into this destination vector created the expression vector for transformation of tobacco chloroplasts with the biolistic method. Correct integration of the transgene into the plastid genome was verified by PCR and the homoplasmic nature of the transformed plants was confirmed by Southern Blot analysis. Expression of the GFP reporter protein was monitored by confocal laser scanning microscopy (CLSM) and quantification by western blot analysis showed a GFP accumulation level of 3% total soluble protein (TSP). The presented results clearly demonstrate that the Gateway(®) recombination sites are compatible with all steps of plastid transformation, from generation of transplastomic plants to expression of GFP. This is the first report of a plastid transformation vector made by the Gateway(®) recombinant cloning technology, which proves the suitability of this system for use in chloroplasts.

Plant-derived vaccines: an approach for affordable vaccines against cervical cancer.

Several types of human papillomavirus (HPV) are causatively associated with cervical cancer, which is the second most common cancer in women worldwide. HPV-16 and 18 are among the high risk types and responsible for HPV infection in more than 70% of the cases. The majority of cervical cancer cases occur in developing countries. Currently available HPV vaccines are expensive and probably unaffordable for most women in low and middle income countries. Therefore, there is a need to develop cost-effective vaccines for these countries. Due to many advantages, plants offer an attractive platform for the development of affordable vaccines. These include low cost of production, scalability, low health risks and the potential ability to be used as unprocessed or partially processed material. Among several techniques, chloroplast transformation is of eminent interest for the production of vaccines because of high yield of foreign protein and lack of transgene transmission through pollen. In this commentary, we focus on the most relevant aspects of plant-derived vaccines that are decisive for the future development of cost-effective HPV vaccines.