Size variations of mesoporous silica nanoparticle control uptake efficiency and delivery of AC2-derived dsRNA for protection against tomato leaf curl New Delhi virus.

KEY MESSAGE: We report the size dependent uptake of dsRNA loaded MSNPs into the leaves and roots of Nicotiana benthamiana plants and accessed for their relative reduction in Tomato leaf curl New Delhi viral load. A non-GMO method of RNA interference (RNAi) has been recently in practice through direct delivery of double stranded RNA into the plant cells. Tomato leaf curl New Delhi virus (ToLCNDV), a bipartitie begomovirus, is a significant viral pathogen of many crops in the Indian subcontinent. Conventional RNAi cargo delivery strategies for instance uses viral vectors and Agrobacterium-facilitated delivery, exhibiting specific host responses from the plant system. In the present study, we synthesized three different sizes of amine-functionalized mesoporous silica nanoparticles (amino-MSNPs) to mediate the delivery of dsRNA derived from the AC2 (dsAC2) gene of ToLCNDV and showed that these dsRNA loaded nanoparticles enabled effective reduction in viral load. Furthermore, we demonstrate that amino-MSNPs protected the dsRNA molecules from nuclease degradation, while the complex was efficiently taken up by the leaves and roots of Nicotiana benthamiana. The real time gene expression evaluation showed that plants treated with nanoparticles of different sizes ~ 10 nm (MSNPDEA), ~ 32 nm (MSNPTEA) and ~ 66 nm (MSNPNH3) showed five-, eleven- and threefold reduction of ToLCNDV in N. benthamiana, respectively compared to the plants treated with naked dsRNA. This work clearly demonstrates the size dependent internalization of amino-MSNPs and relative efficacy in transporting dsRNA into the plant system, which will be useful in convenient topical treatment to protect plants against their pathogens including viruses. Mesoporous silica nanoparticles loaded with FITC, checked for its uptake into Nicotiana benthamiana.

Direct Foliar Application of dsRNA Derived From the Full-Length Gene of NSs of Groundnut Bud Necrosis Virus Limits Virus Accumulation and Symptom Expression.

Groundnut bud necrosis virus (GBNV) is the most significant member of the genus Orthotospovirus occurring in the Indian subcontinent. There is hardly any effective measure to prevent GBNV in crop plants. In order to develop GBNV infection prevention procedure, we examined the effect of the direct foliar application of double-stranded RNA (dsRNA) derived from the full-length NSs gene (1,320 nucleotides) of GBNV. The bacterially expressed dsRNA to the non-structural (dsNSs) gene of GBNV was purified and delivered to plants as an aqueous suspension containing 0.01% Celite for evaluating its efficacy in preventing GBNV infection in systemic host, Nicotiana benthamiana as well as in local lesion and systemic host, cowpea cv. Pusa Komal (Vigna unguiculata). The dsNSs application and challenge-inoculation were conducted in three different combinations, where plants were challenge-inoculated with GBNV a day after, immediately, and a day before the application of dsNSs. N. benthamiana plants, which were not treated with dsRNA showed severe systemic wilting and death by 9-16 days post-inoculation (dpi). The non-treated cowpea plants exhibited many chlorotic and necrotic lesions on the cotyledonary leaves followed by systemic necrosis and death of the plants by 14-16 dpi. The dsNSs treated plants in all the combinations showed significant reduction of disease severity index in both N. benthamiana and cowpea. The treatment combination where the GBNV inoculation was conducted immediately after the dsNSs treatment was found to be the most effective treatment in preventing symptom expression. The viral RNA analysis by real time PCR also showed 20 and 12.5 fold reduction of GBNV in cowpea and N. benthamiana, respectively. Our results suggest that the foliar application of dsRNA derived from the full-length NSs gene of GBNV through Celite is successful in delivering long dsRNA leading to effective prevention of GBNV infection.

Synthetic Biology in Plants, a Boon for Coming Decades.

Recently an enormous expansion of knowledge is seen in various disciplines of science. This surge of information has given rise to concept of interdisciplinary fields, which has resulted in emergence of newer research domains, one of them is 'Synthetic Biology' (SynBio). It captures basics from core biology and integrates it with concepts from the other areas of study such as chemical, electrical, and computational sciences. The essence of synthetic biology is to rewire, re-program, and re-create natural biological pathways, which are carried through genetic circuits. A genetic circuit is a functional assembly of basic biological entities (DNA, RNA, proteins), created using typical design, built, and test cycles. These circuits allow scientists to engineer nearly all biological systems for various useful purposes. The development of sophisticated molecular tools, techniques, genomic programs, and ease of nucleic acid synthesis have further fueled several innovative application of synthetic biology in areas like molecular medicines, pharmaceuticals, biofuels, drug discovery, metabolomics, developing plant biosensors, utilization of prokaryotic systems for metabolite production, and CRISPR/Cas9 in the crop improvement. These applications have largely been dominated by utilization of prokaryotic systems. However, newer researches have indicated positive growth of SynBio for the eukaryotic systems as well. This paper explores advances of synthetic biology in the plant field by elaborating on its core components and potential applications. Here, we have given a comprehensive idea of designing, development, and utilization of synthetic biology in the improvement of the present research state of plant system.

Development of efficient synthetic promoters derived from pararetrovirus suitable for translational research.

MAIN CONCLUSION: In this study, useful hybrid promoters were developed for efficient ectopic gene expression in monocot and dicot plants, and they hold strong prominence in both transgenic research and biotech industries. This study deals with developing novel synthetic promoters derived from Rice Tungro Bacilliform Virus (RTBV) and Mirabilis Mosaic Virus (MMV). Despite numerous availability, there is a severe scarcity of promoters universally suitable for monocot and dicot plants. Here, eight chimeric promoter constructs were synthesized as gBlocks gene fragments through domain swapping and hybridization by incorporating important domains of previously characterized RTBV and MMV promoters. The developed promoter constructs were assessed for transient GUS expression in tobacco protoplast (Xanthi Brad) and agro-infiltrated tobacco, petunia, rice and pearl millet. Protoplast expression analysis showed that two promoter constructs, namely pUPMA-RP1-MP1GUS and pUPMA-RP4-MP1GUS exhibited 3.56 and 2.5 times higher activities than that of the CaMV35S promoter. We had observed the similar type of expression patterns of these promoters in agroinfiltration-based transient studies. RP1-MP1 and RP4-MP1 promoters exhibited 1.87- and 1.68-fold increase expression in transgenic tobacco plants; while, a 1.95-fold increase was found in RP1-MP1 transgenic rice plants when compared their activities with CaMV35S promoter. Furthermore, on evaluating these promoter constructs for their expression in the bacterial system, pUPMA-RP1-MP1GFP was found to have the highest GFP expression. Moreover, the promoter construct was also evaluated for its capacity to express the HMP3 gene. Biobeads of encapsulated bacterial cells expressing HMP3 gene under control of the pUPMA-RP4-MP1 promoter were found to reduce 72.9% copper and 29.2% zinc concentration from wastewater. Our results had demonstrated that the developed promoter constructs could be used for translational research in dicot, monocot plants and bacterial systems for efficient gene expression.

In silico characterization of synthetic promoters designed from mirabilis mosaic virus and rice tungro bacilliform virus.

CaMV35S is the most extensively used promoter for ectopic gene expression in plant system. However, multiple use of this promoter possesses several limitation i.e. homologous based gene silencing and differential suitability in monocot and dicot plants. The strength of a promoter is defined by the presence of cis-acting elements and trans acting nucleic binding factors, thus its strength can be regulated by changing the architecture of these regulatory elements. In the present study, eight hybrid promoters were designed from two parareteroviruses, rice tungro bacilliform viruses (RTBV) and mirabilis mosaic virus (MMV). The eight hybrid promoters, along with parental promoters were characterized for the presence of functional cis-elements and transcription factor binding sites (TFBS), which were predicted using bioinformatics tools such as PLACE and Matinspector. Presence of mirabilis mosaic virus modules for specific functions and over-represented modules was determined using Model inspector. A broad range of cis-elements (85), TFBS (1471) was obtained. Presence of Dehydration responsive element binding factors, Apetala 2 (AP2), WRKY, DNA binding with one finger DOF (DOFF) motifs had shown the functional relevance of these designed promoters with abiotic stress inducibility. In addition to these stress regulating TFBS, the presence of some enhancer like motifs such as P$OCSE, P$TERE, P$TODS, P$ASRC had shown the functional relevance of these promoters as a strong candidate for enhanced expression of ectopic gene.

Fabrication of biobeads expressing heavy metal-binding protein for removal of heavy metal from wastewater.

Heavy metals, being toxic in nature, are one of the most persistent problems in wastewater. Unabated discharge of large amount of heavy metals into water bodies are known to cause several environmental and health impacts. Biological remediation processes like microbial remediation and phytoremediation are proved to be very effective in the reduction of heavy metal pollutants in wastewater. To circumvent the issues involved several peptides and proteins are being explored. Metal-binding capacity, accumulation, and tolerance of heavy metals in bacteria can be upsurge by overexpressing the genes which code for metal-binding proteins. In the present study, an attempt has been made to bioremediate heavy metal toxicity by overexpressing metal-binding proteins. Two expression cassettes harboring top4 metal-binding protein (T4MBP) and human metallothionein 3 (HMP3) were designed under the control of constitutive CaMV 35S promoter and transformed into E.coli TBI cells. E.coli over expressing HMP3 and T4MBP were immobilized in biobeads which were explored for the detoxification of water contaminated with copper and cadmium. Effects on the concentration of heavy metal before and after treatment with beads were estimated with the help of ICP-OES. Noteworthy results were obtained in the case of copper with 87.2% decrease in its concentration after treatment with biobeads. Significant decrement of 32.8% and 27.3% was found in case of zinc and cadmium, respectively. Mechanisms of binding of proteins with heavy metals were further validated by molecular modeling and metal-binding analysis. HMP3 protein was found to be more efficient in metal accumulation as compared with T4MBP. The fabricated biobeads in this study definitely offer an easy and user-handy approach towards the treatment of toxic wastewater.

In silico comparative analysis of promoters derived from plant pararetroviruses.

Promoters are specific sequence of nucleotides present upstream of gene coding region involved in initiation and regulation of transcription. Multiple cis-acting element forms the architecture of promoter to which trans-acting nucleic binding factors bind and regulates its activity. Since 1980 genome of pararetrovirus, are being exploited for developing efficient promoters. Among all of them Cauliflower mosaic virus is the most widely used promoter for gene expression. The basic rational behind the strength of promoter lies in the sequence of cis elements and the spacer nucleotide elements between them, thereby strength of these promoter fragments can be regulated by altering these nucleotide sequences. In the present study sequence of eight putative promoters of plant pararetrovirus are retrieved from National Centre for Biotechnology Information (NCBI) website. These sequence are subjected to various bioinformatics tools comprises of Clustal W, Plant Care, Mathinspector, ModelInspector for establishing the phylogenetic similarity, to identify the quantity and quality of present cis-elements, to find the various common transcription factors binding sites and to determine the presence of module for various specific functions respectively. A range of 28.80-56.0 percentage identification was observed in phylogenetic analysis, with the greatest similarity in Mirabilis mosaic virus and Dahlia mosaic virus. A broad range of cis-elements (51), transcription factor binding site (512) was obtained and 60% observed module are in combination with DOFF motif which shows a function relevance with abiotic stress inducibility. The present study had revealed the functional significance of these elements in gene regulation of pararetrovirus genome and also gives a overall idea for designing novel synthetic promoter.