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.

Isolation of the 3ß-HSD promoter from Digitalis ferruginea subsp. ferruginea and its functional characterization in Arabidopsis thaliana.

BACKGROUND: Although members of the SDR gene family (short chain dehydrogenase) are distributed in kingdom of life, they have diverse roles in stress tolerance mechanism or secondary metabolite biosynthesis. Nevertheless, their precise roles in gene expression or regulation under stress are yet to be understood. METHODS: As a case study, we isolated, sequenced and functionally characterized the 3ß-HSD promoter from Digitalis ferruginea subsp. ferruginea in Arabidopsis thaliana. RESULTS: The promoter fragment contained light and stress response elements such as Box-4, G-Box, TCT-motif, LAMP element, ABRE, ARE, WUN-motif, MYB, MYC, W box, STRE and Box S. The functional analysis of the 3ß-HSD promoter in transgenic Arabidopsis seedlings showed that the promoter was expressed in cotyledon and root elongation zone in 2 days' seedlings. However, this expression was extended to hypocotyl and complete root in 6 days' seedlings. In 20 days-old seedlings, promoter expression was distributed to the whole seedling including hydathodes aperture, vascular bundle, shoot apical meristem, trichomes, midrib, leaf primordia, hypocotyl and xylem tissues. Further, expression of the promoter was enhanced or remained stable under the different abiotic stress conditions like osmotic, heat, cold, cadmium or low pH. In addition, the promoter also showed response to methyl jasmonate (MeJA) application. The expression could not be induced in wounded cotyledon most likely due to lack of interacting elements in the promoter fragment. CONCLUSIONS: Taken together, the 3ß-HSD promoter could be a candidate for the development of transgenic plants especially under changing environmental conditions.

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.

Genetic Diversity, Population Structure and Marker-Trait Association for 100-Seed Weight in International Safflower Panel Using SilicoDArT Marker Information.

Safflower is an important oilseed crop mainly grown in the arid and semi-arid regions of the world. The aim of this study was to explore phenotypic and genetic diversity, population structure, and marker-trait association for 100-seed weight in 94 safflower accessions originating from 26 countries using silicoDArT markers. Analysis of variance revealed statistically significant genotypic effects (p < 0.01), while Turkey samples resulted in higher 100-seed weight compared to Pakistan samples. A Constellation plot divided the studied germplasm into two populations on the basis of their 100-seed weight. Various mean genetic diversity parameters including observed number of alleles (1.99), effective number of alleles (1.54), Shannon's information index (0.48), expected heterozygosity (0.32), and unbiased expected heterozygosity (0.32) for the entire population exhibited sufficient genetic diversity using 12232 silicoDArT markers. Analysis of molecular variance (AMOVA) revealed that most of the variations (91%) in world safflower panel are due to differences within country groups. A model-based structure grouped the 94 safflower accessions into populations A, B, C and an admixture population upon membership coefficient. Neighbor joining analysis grouped the safflower accessions into two populations (A and B). Principal coordinate analysis (PCoA) also clustered the safflower accessions on the basis of geographical origin. Three accessions; Egypt-5, Egypt-2, and India-2 revealed the highest genetic distance and hence might be recommended as candidate parental lines for safflower breeding programs. The mixed linear model i.e., the Q + K model, demonstrated that two DArTseq markers (DArT-45483051 and DArT-15672391) had significant association (p < 0.01) for 100-seed weight. We envisage that identified DArTseq markers associated with 100-seed weight will be helpful to develop high-yielding cultivars of safflower through marker-assisted breeding in the near future.

Expression of ESAT-6 antigen from Mycobacterium tuberculosis in broccoli: An edible plant.

Tuberculosis (TB) is one of the major infectious diseases caused by Mycobacterium tuberculosis. The development of an effective and economical vaccine for controlling TB is essential especially for developing countries. Edible plants can serve as biofactories to produce vaccine antigens. In this study, 6 kDa early secretory antigenic target (ESAT-6) of M. tuberculosis was expressed in Brassica oleracea var. italica via Agrobacterium-mediated transformation to facilitate oral delivery of antigen. ESAT-6 gene was cloned using Gateway® cloning strategy. Transformation and presence of transgene was confirmed through PCR. Expression level of transgene was calculated via quantitative real-time PCR (qRT-PCR) and the maximum integrated transgene number was two. Maximum amount of total soluble fraction of ESAT-6 was evaluated by immunoblotting, estimated to accumulate up to 0.5% of total soluble protein. The recombinant ESAT-6 protein was further purified and detected using silver staining and Western blotting. ESAT-6 protein induced humoral immune response in mice immunized orally and subcutaneously. The expression of M. tuberculosis antigen in edible plants could aid in the development of cost-effective and oral delivery of an antigen-based subunit vaccine against TB. To the best our knowledge, it is the first report of expression of a vaccine antigen in broccoli.

Suppressing photorespiration for the improvement in photosynthesis and crop yields: A review on the role of S-allantoin as a nitrogen source.

Environmental variations resulting in biotic and abiotic stresses demand adaptive changes in the photosynthetic machinery. To cope with these challenges, plant scientists are constantly striving to enhance photosynthetic activity. The photorespiration pathway, which fixes O2 and releases CO2 in C3 plants, competes with photosynthesis. One method to increase yield would be to enhance photosynthesis by engineering the photorespiratory pathway. To date, three engineered photorespiratory pathways have been produced, of which two have been proven experimentally in the model plant, Arabidopsis thaliana. These approaches might be helpful in enhancing crop resilience to future environmental challenges. In partially photorespiratory suppressed plants, it is hypothesized that a gene cluster may have formed between bacterial glycolate dehydrogenase (GDH), glyoxylate carboligase (GCL), and tartronic semi aldehyde (TSR) genes with Arabidopsis allantoin degradation genes like Arabidopsis allantoinase (AtALN) to utilize S-allantoin as a source of nitrogen. Observations of the use of allantoin as an exclusive source of nitrogen or energy by Arabidopsis and Escherichia coli led us to propose a genetic switch control model between nitrogen assimilation and energy producing pathways in partially photorespiratory suppressed plants.

Uncovering Phenotypic Diversity and DArTseq Marker Loci Associated with Antioxidant Activity in Common Bean.

Antioxidants play an important role in animal and plant life owing to their involvement in complex metabolic and signaling mechanisms, hence uncovering the genetic basis associated with antioxidant activity is very important for the development of improved varieties. Here, a total of 182 common bean (Phaseolus vulgaris) landraces and six commercial cultivars collected from 19 provinces of Turkey were evaluated for seed antioxidant activity under four environments and two locations. Antioxidant activity was measured using ABTS radical scavenging capacity and mean antioxidant activity in common bean landraces was 20.03 µmol TE/g. Analysis of variance reflected that genotype by environment interaction was statistically non-significant and heritability analysis showed higher heritability of antioxidant activity. Variations in seed color were observed, and a higher antioxidant activity was present in seeds having colored seed as compared to those having white seeds. A negative correlation was found between white-colored seeds and antioxidant activity. A total of 7900 DArTseq markers were used to explore the population structure that grouped the studied germplasm into two sub-populations on the basis of their geographical origins and trolox equivalent antioxidant capacity contents. Mean linkage disequilibrium (LD) was 54%, and mean LD decay was 1.15 Mb. Mixed linear model i.e., the Q + K model demonstrated that four DArTseq markers had significant association (p < 0.01) for antioxidant activity. Three of these markers were present on chromosome Pv07, while the fourth marker was located on chromosome Pv03. Among the identified markers, DArT-3369938 marker showed maximum (14.61%) variation. A total of four putative candidate genes were predicted from sequences reflecting homology to identified DArTseq markers. This is a pioneering study involving the identification of association for antioxidant activity in common bean seeds. We envisage that this study will be very helpful for global common bean breeding community in order to develop cultivars with higher antioxidant activity.

CRISPR/Cas9-Mediated Immunity in Plants Against Pathogens.

Global crop production is highly threatened due to pathogen invasion. The huge quantity of pesticides application, although harmful to the environment and human health, is carried out to prevent the crop losses worldwide, every year. Therefore, understanding the molecular mechanisms of pathogenicity and plant resistance against pathogen is important. The resistance against pathogens is regulated by three important phytohormones viz. salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). Here we review possible role of CRISPR technology to understand the plant pathogenicity by mutating genes responsible for pathogen invasion or up-regulating the phytohormones genes or resistant genes. Thus hormone biosynthesis genes, receptor and feeding genes of pathogens could be important targets for modifications using CRISPR/Cas9 following multiplexing tool box strategy in order to edit multiple genes simultaneously to produce super plants. Here we put forward our idea thatthe genes would be either mutated in case of plant receptor protein targets of pathogens or up-regulation of resistant genes or hormone biosynthesis genes will be better choice for resistance against pathogens.

An Overview of Cardenolides in Digitalis - More Than a Cardiotonic Compound.

The genus Digitalis L. containing species, commonly known as the "foxglove", is the main source of cardenolides, which have various pharmacological properties effective against certain pathological conditions including myocardial infarction, arterial hypertension, cardiac dysfunction, angina, and hypertrophy. Togehter with a prime effect of controlling the heart rhythm, many workers demonstrated that lanatoside C and some other cardiac glycosides are effective in several cancer treatments such as prostate and breast cancers. Due to digoxigenin derivatives of cardenolides, which are mainly used for medicinal purposes, such as digoxigenin, D. lanata as a main source is of great interest for commercial scale production of cardenolides in Europe. Phytochemical studies on cardenolides, naturally occurring plant secondary metabolites, have mainly focused on the species of the genus Digitalis L., as the members of this family have a high level and diverse content of cardenolides. During the last few decades, plant tissue culture techniques have been optimised for many plant species including Digitalis, however, the production capacity of cardenolides somehow failed to reach a commercially desired extent. In this review paper, the genus Digitalis is evaluated in terms of its main botanical and physiological features, traditional uses, molecular genetics and metabolomics, cellular mechanism of action, medicinal uses, clinical pharmacology, drug interactions, therapy in the management of cardiovascular disorders, potential utility of therapy in extracardiac conditions, and toxicity.

Comparative Transcriptional Profiling of Primed and Non-primed Rice Seedlings under Submergence Stress.

Submergence stress is a limiting factor for direct-seeded rice systems in rainfed lowlands and flood-prone areas of South and Southeast Asia. The present study demonstrated that submergence stress severely hampered the germination and seedling growth of rice, however, seed priming alleviated the detrimental effects of submergence stress. To elucidate the molecular basis of seed priming-induced submergence tolerance, transcriptome analyses were performed using 4-day-old primed (selenium-Se and salicylic acid-SA priming) and non-primed rice seedlings under submergence stress. Genomewide transcriptomic profiling identified 2371 and 2405 transcripts with Se- and SA-priming, respectively, that were differentially expressed in rice compared with non-priming treatment under submergence. Pathway and gene ontology term enrichment analyses revealed that genes involved in regulation of secondary metabolism, development, cell, transport, protein, and metal handling were over-represented after Se- or SA-priming. These coordinated factors might have enhanced the submergence tolerance and maintained the better germination and vigorous seedling growth of primed rice seedlings. It was also found that many genes involved in cellular and metabolic processes such as carbohydrate metabolism, cellular, and metabolic biosynthesis, nitrogen compound metabolic process, transcription, and response to oxidative stress were induced and overlapped in seed priming treatments, a finding which reveals the common mechanism of seed priming-induced submergence tolerance. Taken together, these results may provide new avenues for understanding and advancing priming-induced responses to submergence tolerance in crop plants.

Need of cost-effective vaccines in developing countries: What plant biotechnology can offer?

To treat current infectious diseases, different therapies are used that include drugs or vaccines or both. Currently, the world is facing an increasing problem of drug resistance from many pathogenic microorganisms. In majority of cases, when vaccines are used, formulations consist of live attenuated microorganisms. This poses an additional risk of infection in immunocompromised patients and people suffering from malnutrition in developing countries. Therefore, there is need to improve drug therapy as well as to develop next generation vaccines, in particular against infectious diseases with highest mortality rates. For patients in developing countries, costs related to treatments are one of the major hurdles to reduce the disease burden. In many cases, use of prophylactic vaccines can help to control the incidence of infectious diseases. In the present review, we describe some infectious diseases with high impact on health of people in low and middle income countries. We discuss the prospects of plants as alternative platform for the development of next-generation subunit vaccines that can be a cost-effective source for mass immunization of people in developing countries.

Recent developments in therapeutic protein expression technologies in plants.

Infectious diseases and cancers are some of the commonest causes of deaths throughout the world. The previous two decades have witnessed a combined endeavor across various biological sciences to address this issue in novel ways. The advent of recombinant DNA technologies has provided the tools for producing recombinant proteins that can be used as therapeutic agents. A number of expression systems have been developed for the production of pharmaceutical products. Recently, advances have been made using plants as bioreactors to produce therapeutic proteins directed against infectious diseases and cancers. This review highlights the recent progress in therapeutic protein expression in plants (stable and transient), the factors affecting heterologous protein expression, vector systems and recent developments in existing technologies and steps towards the industrial production of plant-made vaccines, antibodies, and biopharmaceuticals.