Novel approaches to circumvent the devastating effects of pests on sugarcane.

Sugarcane (Saccharum officinarum L.) is a cash crop grown commercially for its higher amounts of sucrose, stored within the mature internodes of the stem. Numerous studies have been done for the resistance development against biotic and abiotic stresses to save the sucrose yields. Quality and yield of sugarcane production is always threatened by the damages of cane borers and weeds. In current study two problems were better addressed through the genetic modification of sugarcane for provision of resistance against insects and weedicide via the expression of two modified cane borer resistant CEMB-Cry1Ac (1.8 kb), CEMB-Cry2A (1.9 kb) and one glyphosate tolerant CEMB-GTGene (1.4 kb) genes, driven by maize Ubiquitin Promoter and nos terminator. Insect Bio-toxicity assays were carried out for the assessment of Cry proteins through mortality percent of shoot borer Chilo infuscatellus at 2nd instar larvae stage. During V0, V1 and V2 generations young leaves from the transgenic sugarcane plants were collected at plant age of 20, 40, 60, 80 days and fed to the Chilo infuscatellus larvae. Up to 100% mortality of Chilo infuscatellus from 80 days old transgenic plants of V2 generation indicated that these transgenic plants were highly resistant against shoot borer and the gene expression level is sufficient to provide complete resistance against target pests. Glyphosate spray assay was carried out for complete removal of weeds. In V1-generation, 70-76% transgenic sugarcane plants were found tolerant against glyphosate spray (3000 mL/ha) under field conditions. While in V2-generation, the replicates of five selected lines 4L/2, 5L/5, 6L/5, L8/4, and L9/6 were found 100% tolerant against 3000 mL/ha glyphosate spray. It is evident from current study that CEMB-GTGene, CEMB-Cry1Ac and CEMB-Cry2A genes expression in sugarcane variety CPF-246 showed an efficient resistance against cane borers (Chilo infuscatellus) and was also highly tolerant against glyphosate spray. The selected transgenic sugarcane lines showed sustainable resistance against cane borer and glyphosate spray can be further exploited at farmer's field level after fulfilling the biosafety requirements to boost the sugarcane production in the country.

The role of plant expression platforms in biopharmaceutical development: possibilities for the future.

Introduction: Plant-made vaccines have been in the pipeline for nearly thirty years. Generated stably in transgenic plants or transiently using virus expression systems, pharmaceuticals have been developed to address global pandemics as well as several emerging One Health Diseases.Areas covered: This review describes the generation of plant-made vaccines to address some of the world's most growing health concerns, including both infectious and non-communicable diseases, such as cancer. The review provides an overview of the research taking place in this field over the past three to five years. The PubMed database was searched under the topic of plant-made vaccine between the periods of 2014 and 2019.Expert opinion: While vaccines and other biologics have been shown to be cheap safe and efficacious, they have not yet entered the marketplace largely due to regulatory constraints. The lack of an appropriate regulatory structure to guide plant-made vaccines through to commercial development has stalled efforts to provide life-saving medicines to low- and middle-income families. In my opinion, it is paramount that regulatory hurdles are mitigated to address emerging infectious diseases such as Ebola and Zika in a timely manner.

Repurposing Plant Virus Nanoparticles.

Plants have been explored for many years as inexpensive and versatile platforms for the generation of vaccines and other biopharmaceuticals. Plant viruses have also been engineered to either express subunit vaccines or act as epitope presentation systems. Both icosahedral and helical, filamentous-shaped plant viruses have been used for these purposes. More recently, plant viruses have been utilized as nanoparticles to transport drugs and active molecules into cancer cells. The following review describes the use of both icosahedral and helical plant viruses in a variety of new functions against cancer. The review illustrates the breadth of variation among different plant virus nanoparticles and how this impacts the immune response.

Recent Patents in Oncolytic Virotherapy.

Recent innovative and advanced developments in the diagnosis and treatment of human diseases as well as enhanced in-depth understanding of virus molecular biology have opened novel avenues with respect to the patent landscape. Included are viruses utilized in the development of anticancer agents, agents that are employed against the spread of infectious viral diseases, RNA silencing agents and virus-derived expression vectors that can be used for over-expression of therapeutic proteins or as gene therapy vehicles. The current review describes several recent patents pertaining to virus sequences and their medical and biotechnological applications.

Virus expression vectors.

For many years now, virus expression vectors have been explored as a mechanism for gene delivery, cancer therapy and vaccine development. More recently, the next generation of virus vectors have been generated that possess greater attributes such as tissue specificity and improved expression levels, while at the same time lack many of the shortcomings of their predecessors, such as issues regarding immunogenicity and safety. This review article describes several of the recent patents that have been issued in the field of virus expression vector development. Innovations in both plant and animal virus expression vectors are covered. The review concludes with a discussion of future prospects of virus expression vectors as tools in medical research.

Recent patents involving virus nucleotide sequences; host defense, RNA silencing and expression vector strategies.

Improved knowledge of the molecular biology of viruses, including recent gains in virus sequence data analysis, has greatly contributed to recent innovations in medical diagnostics, therapeutics, drug development and other related areas. Virus sequences have been used for the development of vaccines and antiviral agents to block the spread of viral infections, as well as to target and battle chronic diseases such as cancer. Virus sequences are now routinely employed in a wide array of RNA silencing technologies. Viruses can also be engineered into expression vectors which in turn can be used as protein production platforms as well as delivery vehicles for gene therapies. This review article outlines a number of patents that have been recently issued with respect to virus sequence data and describes some of their biotechnological applications.

Innovations in siRNA research: a technology comes of age.

Short interfering RNAs, or siRNAs, belong to a class of RNA species which play a role in both cellular defence and gene regulation. siRNAs comprise a larger portion of the RNA interference pathway that includes the degradation of RNAs which possess complementarily to specific target sequences. This property has given siRNA technology the potential to become a powerful new tool for a wide variety of disciplines, ranging from the design of novel anti-cancer agents to applications in agriculture. The following review outlines patents that have been issued over the past 6 months concerning siRNA technology. Patents are discussed which encompass improved delivery systems for cellular uptake of siRNAs, new therapeutics to combat human diseases, and unique uses of siRNAs to advance plant science. The review also provides detailed lists of the most recent patents that have been issued which cover these areas of siRNA technology, and paves the way for future innovations based on RNA interference in the life sciences.

Amplification of genome-integrated BeYDV replicons by transient expression of Rep in Arabidopsis thaliana

Bean yellow dwarf geminivirus (BeYDV) has been used as a potential vector to improve foreign gene expression, specifically, to achieve higher yields of vaccine proteins in plants. Previously, we have shown that when the BeYDV replication initiator protein Rep was provided in trans, replication and gene expression of GUS were enhanced enormously from a BeYDV expression vector in a transient assay system. In this paper, transgenic lines of Arabidopsis (cv. Columbia) were generated harboring the BeYDV cis-acting elements required for replication. Constructs encoding BeYDV Rep or intronless Rep open reading frames (ORFs) were transiently introduced into transgenic plants via Agrobacterium-mediated infiltration in order to examine the relative levels of replication and expression of the genome-integrated GUS reporter gene. This study shows that expression of Rep protein was regulated in trans from a separate cassette which enabled the rescue, replication and enhancement of the genome-integrated GUS gene in transgenic Arabidopsis. We conclude that Rep expression can be effectively controlled in Arabidopsis plants, and that regulation of Rep expression can result in the amplification of a genome-integrated foreign gene by circumventing the negative effects of gene silencing.

Expression and mutational analysis of Autographa californica nucleopolyhedrovirus HCF-1: functional requirements for cysteine residues.

The host cell-specific factor 1 gene (hcf-1) of the baculovirus Autographa californica multiple nucleopolyhedrovirus is required for efficient virus growth in TN368 cells but is dispensable for virus replication in SF21 cells. However, the mechanism of action of hcf-1 is unknown. To begin to understand its function in virus replication we have investigated the expression and localization pattern of HCF-1 in infected cells. Analysis of virus-infected TN368 cells showed that hcf-1 is expressed at an early time in the virus life cycle, between 2 and 12 h postinfection, and localized the protein to punctate nuclear foci. Through coprecipitation experiments we have confirmed that HCF-1 self-associates into dimers or higher-order structures. We also found that overexpression of HCF-1 repressed expression from the hcf-1 promoter in transient reporter assays. Mutagenesis of cysteine residues within a putative RING finger domain in the amino acid sequence of HCF-1 abolished self-association activity and suggests that the RING domain may be involved in this protein-protein interaction. A different but overlapping set of cysteine residues were required for efficient gene repression activity. Functional analysis of HCF-1 mutants showed that the cysteine amino acids required for both self-association and gene repression activities of HCF-1 were also required for efficient late-gene expression and occlusion body formation in TN368 cells. Mutational analysis also identified essential charged and hydrophobic amino acids located between two of the essential cysteine residues. We propose that HCF-1 is a RING finger-containing protein whose activity requires HCF-1 self-association and gene repression activity.

Expression of a vaccine protein in a plant cell line using a geminivirus-based replicon system.

Edible vaccines have been generated from both transgenic plants as well as from plant viral vectors. Here, we have taken the best attributions of both systems and designed a minimalized version of the bean yellow dwarf geminivirus (BeYDV)-based replicon consisting of the cis-acting elements required for BeYDV replication as a means to express foreign genes in a plant cell line. Replication can be switched on at high levels upon expression of the BeYDV Rep protein, and gene expression enhanced enormously. Construction of an expression cassette encoding a synthetic vaccine gene and analysis of expression levels of a vaccine protein in a plant cell line system are described.

Independent expression of Rep and RepA and their roles in regulating bean yellow dwarf virus replication.

Bean yellow dwarf virus (BeYDV) is a mastrevirus specific for dicotyledenous hosts. It contains four ORFs encoding a movement protein, a coat protein, and two Rep gene products, Rep and RepA, which are encoded by two overlapping ORFs. In this study, the roles of Rep and RepA in regulating replication of the BeYDV-based replicon were investigated by uncoupling them and placing Rep and RepA each under constitutive promoter control. Constitutive expression of both Rep and RepA supported replication and enhanced gene expression. When a reporter plasmid containing the Rep gene in the context of its native promoter was supplemented with additional Rep protein, replication was enhanced but the increase in gene expression was found to be more modest. Furthermore, expression of constitutively expressed RepA alone was found to reduce replication of this reporter construct as well as delay BeYDV replication in general. The effect of a RepA mutant with an altered retinoblastoma-related-protein binding motif on the efficiency of BeYDV replication was also examined. This mutant was found to severely diminish replication efficiency. Finally, the relationship of BeYDV coat protein to virus replication and reporter gene expression was investigated. Addition of coat protein increased accumulation of single-stranded DNA and had a detrimental effect on reporter gene expression.