Plant Virus-based Vectors Research Articles

Plant virology in the 21st century in China: Recent advances and future directions.

Plant viruses are a group of intracellular pathogens that persistently threaten global food security. Significant advances in plant virology have been achieved by Chinese scientists over the last 20 years, including basic research and technologies for preventing and controlling plant viral diseases. Here, we review these milestones and advances, including the identification of new crop-infecting viruses, dissection of pathogenic mechanisms of multiple viruses, examination of multilayered interactions among viruses, their host plants, and virus-transmitting arthropod vectors, and in-depth interrogation of plant-encoded resistance and susceptibility determinants. Notably, various plant virus-based vectors have also been successfully developed for gene function studies and target gene expression in plants. We also recommend future plant virology studies in China.

A cowpea severe mosaic virus-based vector simplifies virus-induced gene silencing and foreign protein expression in soybean

BackgroundSoybean gene functions cannot be easily interrogated through transgenic disruption (knock-out) of genes-of-interest, or transgenic overexpression of proteins-of-interest, because soybean transformation is time-consuming and technically challenging. An attractive alternative is to administer transient gene silencing or overexpression with a plant virus-based vector. However, existing virus-induced gene silencing (VIGS) and/or overexpression vectors suitable for soybean have various drawbacks that hinder their widespread adoption.ResultsWe describe the development of a new vector based on cowpea severe mosaic virus (CPSMV), a plus-strand RNA virus with its genome divided into two RNA segments, RNA1 and RNA2. This vector, designated FZ, incorporates a cloning site in the RNA2 cDNA, permitting insertion of nonviral sequences. When paired with an optimized RNA1 construct, FZ readily infects both Nicotiana benthamiana and soybean. As a result, FZ constructs destined for soybean can be first delivered to N. benthamiana in order to propagate the modified viruses to high titers. FZ-based silencing constructs induced robust silencing of phytoene desaturase genes in N. benthamiana, multiple soybean accessions, and cowpea. Meanwhile, FZ supported systemic expression of fluorescent proteins mNeonGreen and mCherry in N. benthamiana and soybean. Finally, FZ-mediated expression of the Arabidopsis transcription factor MYB75 caused N. benthamiana to bear brown leaves and purple, twisted flowers, indicating that MYB75 retained the function of activating anthocyanin synthesis pathways in a different plant.ConclusionsThe new CPSMV-derived FZ vector provides a convenient and versatile soybean functional genomics tool that is expected to accelerate the characterization of soybean genes controlling crucial productivity traits.

A Versatile Expression Platform in Insects and Cereals Based on a Cytorhabdovirus.

In recent years, plant virus-based vectors have been widely applied to express heterologous proteins for genomic studies and commercial production. Among these versatile RNA viral vectors, the barley yellow striate mosaic virus (BYSMV)-based expression vector system has outstanding capability to express large and multiple heterologous proteins. Here we describe a detailed protocol for expression of heterologous proteins using BYSMV expression systems in monocot plants and insects.

Plant Virus-Derived Vectors: Applications in Agricultural and Medical Biotechnology.

Major advances in our understanding of plant viral genome expression strategies and the interaction of a virus with its host for replication and movement, induction of disease, and resistance responses have been made through the generation of infectious molecules from cloned viral sequences. Autonomously replicating viral vectors derived from infectious clones have been exploited to express foreign genes in plants. Applications of virus-based vectors include the production of human/animal therapeutic proteins in plant cells and the specific study of plant biochemical processes, including those that confer resistance to pathogens. Additionally, virus-induced gene silencing, which is RNA mediated and triggered through homology-dependent RNA degradation mechanisms, has been exploited as an efficient method to study the functions of host genes in plants and to deliver small RNAs to insects. New and exciting strategies for vector engineering, delivery, and applications of plant virus-based vectors are the subject of this review.

A Plant Virus-Based Vector System for Gene Function Studies in Pepper.

While pepper (Capsicum annuum) is a highly recalcitrant species for genetic transformation studies, plant virus-based vectors can provide alternative and powerful tools for transient regulation and functional analysis of genes of interest in pepper. In this study, we established an effective virus-based vector system applicable for transient gain- and loss-of-function studies in pepper using Broad bean wilt virus2 (BBWV2). We engineered BBWV2 as a dual gene expression vector for simultaneous expression of two recombinant proteins in pepper cells. In addition, we established enhanced and stable expression of recombinant proteins from the BBWV2-based dual vector via coexpression of a heterologous viral suppressor of RNA silencing. We also developed a BBWV2-based virus-induced gene silencing (VIGS) vector, and we successfully silenced the phytoene desaturase gene (PDS) using the BBWV2-based VIGS vector in various pepper cultivars. Additionally, we optimized the BBWV2-based VIGS system in pepper by testing the efficiency of PDS gene silencing under different conditions. This BBWV2-based vector system represents a convenient approach for rapid and simple analysis of gene functions in pepper.

Development of Soybean Yellow Mottle Mosaic Virus-Based Expression Vector for Heterologous Protein Expression in French Bean.

Plant virus-based vectors provide attractive and valuable tools for rapid production of recombinant protein in large quantities as they produce systemic infections in differentiated plant tissues. In the present study, we engineered the Soybean yellow mottle mosaic virus (SYMMV) as a gene expression vector which is a promising candidate for systemic expression of foreign proteins in French bean plants. Full virus vector strategy was exploited for insertion of foreign gene by inserting MCS through PCR in the circular pJET-SYMMV clone. To examine the ability of the SYMMV vector system, GFP gene was cloned after the start codon of coat protein (CP) so that its expression was driven by the SYMMV-CP subgenomic promoter. When in vitro run off SYMMV-GFP transcript was mechanically inoculated to French bean leaves, good level of GFP expression was observed through confocal microscopy up to 40 dpi. Expression of heterologous protein was also confirmed through ISEM, DAC-ELISA and RT-PCR with specific primers at 20dpi. The recombinant SYMMV construct was stable in in vitro runoff transcript inoculated plants but the inserted GFP was lost in progeny virion inoculated plants. The system developed here will be useful for further studies of SYMMV gene functions and exploitation of SYMMV as a gene expression vector.

Integration of Cell-Penetrating Peptides with Rod-like Bionanoparticles: Virus-Inspired Gene-Silencing Technology.

Inspired by the high gene transfer efficiency of viral vectors and to avoid side effects, we present here a 1D rod-like gene-silencing vector based on a plant virus. By decorating the transacting activator of transduction (TAT) peptide on the exterior surface, the TAT-modified tobacco mosaic virus (TMV) achieves a tunable isoelectric point (from ∼3.5 to ∼9.6) depending on the TAT dose. In addition to enhanced cell internalization, this plant virus-based vector (TMV-TAT) acquired endo/lysosomal escape capacity without inducing lysosomal damage, resulting in both high efficiency and low cytotoxicity. By loading silencer green fluorescent protein (GFP) siRNA onto the TMV-TAT vector (siRNA@TMV-TAT) and interfering with GFP-expressing mouse epidermal stem cells (ESCs/GFP) in vitro, the proportion of GFP-positive cells could be knocked down to levels even lower than 15% at a concentration of ∼100% cell viability. Moreover, by interfering with GFP-expressing highly metastatic hepatocellular carcinoma (MHCC97-H/GFP) tumors in vivo, treatment with siRNA@TMV-TAT complexes for 10 days achieved a GFP-negative rate as high as 80.8%. This work combines the high efficiency of viral vectors and the safety of nonviral vectors and may provide a promising strategy for gene-silencing technology.

Efficient Protein Expression and Virus-Induced Gene Silencing in Plants Using a Crinivirus-Derived Vector.

Plant virus-based vectors are valuable tools for recombinant gene expression and functional genomics for both basic and applied research. In this study, Lettuce infectious yellows virus (LIYV) of the genus Crinivirus was engineered into a virus vector that is applicable for efficient protein expression and virus-induced gene silencing (VIGS) in plants. We examined gene replacement and “add a gene” strategies to develop LIYV-derived vectors for transient expression of the green fluorescent protein (GFP) reporter in Nicotiana benthamiana plants. The latter yielded higher GFP expression and was further examined by testing the effects of heterologous controller elements (CEs). A series of five vector constructs with progressively extended LIYV CP sgRNA CEs were tested, the longest CE gave the highest GFP expression but lower virus accumulation. The whitefly transmissibility of the optimized vector construct to other host plants, and the capability to accommodate and express a larger gene, a 1.8 kb β-glucuronidase (GUS) gene, were confirmed. Furthermore, the LIYV vector was also validated VIGS by silencing the endogenous gene, phytoene desaturase (PDS) in N. benthamiana plants, and the transgene GFP in N. benthamiana line 16c plants. Therefore, LIYV-derived vectors could provide a technical reference for developing vectors of other economically important criniviruses.

Vector construction to enhance GP5 antigen expression of virus PRRS in plant cells

Plant vaccine is a new tool to enrich available vaccine resources for protection of human and animal health. Plant vaccine offers several advantages over current conventional vaccines, including that storage and transportationare convenient after lyophilization, production costs are low, and the contamination of mammalian pathogens is avoided. However, the low expression of foreign genes in plant still exists that limits the application of this kind of vaccine. Presently, plant virus-based expression vectors represent a technology that enables high levels of recombination proteins to be produced efficiently in plant cells. In this study, a modified TMV-based vector to elevate glycoprotein GP5 of PRRSV in plant cells without negative effects in the development of plant cells was developed. Gene encoded GP5 was replaced CP of TMV and the whole was regulated by the heat shock protein Hsp 18.2 promoter. Transgenic BY-2 cells carrying pHsp-TMV-GP5 showed the normal development compared to the ones harbouring pCB-35S-TMV-GP5. The results showed that the Hsp-TMV-GP5 construct do not have a negative effect on viability of transgenic plant cells. A further study will be carried out to investigate the expression of GP5 in stable transgenic plant cells to confirm the benefit of the modified expression vector in development of rapid and cost-effective antigens in plant cell suspension culture.

Gene silencing and gene expression in phytopathogenic fungi using a plant virus vector

RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.

Monoclonal Antibody Purification (Nicotiana benthamiana Plants).

Plant-based expression systems provide an alternative biomanufacturing platform for recombinant proteins (Matoba et al., 2011). In particular, plant virus-based vectors can overexpress proteins within days in the leaf tissue of Nicotiana benthamiana (N. benthamiana). To overcome the issues of genetic instability and limited infectivity of recombinant viruses, Agrobacterium-mediated delivery of "deconstructed" virus vectors has become the mainstay for the production of large and/or multicomponent proteins, such as immunoglobulin (Ig)G monoclonal antibodies (mAbs). Here, we describe a method of producing human IgG mAbs in N. benthamiana using the tobamoviral replicon vector magnICON®. The vector can express up to a few hundred mg of a mAb per kg of leaf material in 7 days. A representative case for the broadly neutralizing anti-HIV and anti-influenza mAbs, VRC01 and CR6261 respectively, is shown (Hamorsky et al., 2013). Leaf tissue is homogenized and the extract is clarified by filtration and centrifugation. The mAb is purified by fast protein liquid chromatography (FPLC) using Protein A affinity and Phenyl HP hydrophobic interection resins.

A dual gene‐silencing vector system for monocot and dicot plants

Plant virus-based gene-silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus-induced gene-silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene-silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N.benthamiana. GFP transgenic plants co-agro-inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat-shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV- but not BaMV-based vector could enhance gene-silencing efficiency in newly emerging leaves of N.benthamiana deficient in RNA-dependant RNA polymerase 6. The dual gene-silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.

Induction of protective immunity in chickens immunized with plant-made chimeric Bamboo mosaic virus particles expressing very virulent Infectious bursal disease virus antigen

Very virulent Infectious bursal disease virus (vvIBDV) causes a highly contagious disease in young chickens and leads to significant economic loss in the poultry industry. Effective new vaccines are urgently needed. Autonomously replicating plant virus-based vector provides attractive means for producing chimeric virus particles (CVPs) in plants that can be developed into vaccines. In this study, we demonstrate the potential for vaccine development of Bamboo mosaic virus (BaMV) epitope-presentation system, where the antigen from vvIBDV VP2 was fused to the N-terminus of BaMV coat protein. Accordingly, an infections plasmid, pBIBD2, was constructed. Inoculation of the recombinant BaMV clone pBIBD2 enabled the generation of chimeric virus, BIBD2, and stable expression of IBDV VP2 antigen on its coat protein. After intramuscular immunization with BIBD2 CVPs, chickens produced antibodies against IBDV and were protected from vvIBDV (V263/TW strain) challenges. These results corroborate the feasibility of BaMV-based CVP platform in plants for the development and production of vaccines against IBDV.

Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression

We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein expression, based on Alternanthera mosaic virus (AltMV), for infection of a wide range of host plants including Nicotiana benthamiana and Arabidopsis thaliana by either mechanical inoculation of in vitro transcripts or via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection from a binary vector in the absence of the Cauliflower mosaic virus 35S promoter. An artificial bipartite viral vector delivery system was created by separating the AltMV RNA-dependent RNA polymerase and Triple Gene Block (TGB)123-Coat protein (CP) coding regions into two constructs each bearing the AltMV 5' and 3' non-coding regions, which recombined in planta to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and equivalent changes in other potexvirus TGB1 proteins, affected RNA silencing suppression efficacy and suitability of the vectors from protein expression to VIGS.

The development of an efficient multipurpose bean pod mottle virus viral vector set for foreign gene expression and RNA silencing.

Plant viral vectors are valuable tools for heterologous gene expression, and because of virus-induced gene silencing (VIGS), they also have important applications as reverse genetics tools for gene function studies. Viral vectors are especially useful for plants such as soybean (Glycine max) that are recalcitrant to transformation. Previously, two generations of bean pod mottle virus (BPMV; genus Comovirus) vectors have been developed for overexpressing and silencing genes in soybean. However, the design of the previous vectors imposes constraints that limit their utility. For example, VIGS target sequences must be expressed as fusion proteins in the same reading frame as the viral polyprotein. This requirement limits the design of VIGS target sequences to open reading frames. Furthermore, expression of multiple genes or simultaneous silencing of one gene and expression of another was not possible. To overcome these and other issues, a new BPMV-based vector system was developed to facilitate a variety of applications for gene function studies in soybean as well as in common bean (Phaseolus vulgaris). These vectors are designed for simultaneous expression of multiple foreign genes, insertion of noncoding/antisense sequences, and simultaneous expression and silencing. The simultaneous expression of green fluorescent protein and silencing of phytoene desaturase shows that marker gene-assisted silencing is feasible. These results demonstrate the utility of this BPMV vector set for a wide range of applications in soybean and common bean, and they have implications for improvement of other plant virus-based vector systems.

Plant-produced human growth hormone shows biological activity in a rat model

Plants have been shown to be efficient systems for expressing a wide range of recombinant proteins from various origins. Here, using a plant virus-based expression vector to produce human growth hormone (hGH) in Nicotiana benthamiana plants, we demonstrate, for the first time, that the plant-produced hGH (pphGH) is biologically active in a hypophysectomized rat model. We observed an average weight gain of approximately 17 g per animal in a group of 10 animals that were injected subcutaneously with pphGH with 60 microg/dose for 10 days. With the increasing demand for hGH, accompanied with the need to make this recombinant protein available to a wider population at a more reasonable cost, plants provide a feasible alternative to current production platforms.

Systemic gene delivery into soybean by simple rub-inoculation with plasmid DNA of a Soybean mosaic virus-based vector

Plant virus-based vectors provide attractive and valuable tools for conventional transgenic technology and gene function studies in plants. In the present study, we established the infectivity of intact plasmid DNA of Soybean mosaic virus (SMV) cDNA upon simple rub-inoculation of soybean leaves by utilizing viral transcription and processing signals to produce infectious in vivo transcripts. Furthermore, we engineered this SMV cDNA clone as a gene delivery vector for systemic expression of foreign proteins in soybean. Using this SMV-based vector, several genes with different biological activities were successfully expressed and stably maintained following serial plant passage in soybean. Thus, DNA-mediated gene delivery using this SMV-based vector provides a rapid and cost-effective approach for the overproduction of valuable proteins and for the evaluation of new traits in soybean after simple rub-inoculation onto leaves.

Recent Developments in Design and Application of Plant Virus-based Gene Vectors

A new horizon is lit up by exploiting plant viruses as the vectors to deliver foreign genes into plants for various purposes such as production of valuable pharmaceutical proteins, to understand pathogenesis of a plant virus, and to establish gene silencing for blocking, for example, production of an undesirable intermediate metabolite in a metabolic pathway. Here, the recent patents concerning the design of new gene vectors on the basis of the genomes of the viruses including Tobacco mosaic virus (TMV), Potato virus X (PVX), Tobacco rattle virus (TRV), Cowpea mosaic virus (CPMV), Bean Yellow dwarf virus (BeYDV), Beet soil-borne mosaic virus (BSbMV) and Potato mop top virus (PMTV) will be reviewed. Also, applications of the recently designed vectors for production of foreign proteins such as human lysosomal enzymes expressed from TMV-based vectors, human and animal viral vaccines expressed from CPMV-based vectors and glucose oxidase from BSbMV-based vector will be reported. In addition, gene silencing capabilities of the vectors based on PVX and the seed coating gene delivery system will be considered.

Transient expression of the ectodomain of matrix protein 2 (M2e) of avian influenza A virus in plants

We have previously reported an expression system based on the capsid protein gene (CP) of cucumber mosaic virus (CMV) placed under transcriptional control of a potato virus X (PVX)-based vector. PVX-expressed CMV CP formed virus-like particles, which served as carriers for heterologous antigens of the Newcastle disease virus (NDV). In this work, we applied our expression tool toward the development of plant-derived vaccine candidate against avian influenza A virus. Twenty-three amino acid-long extracellular domain of the viral M2 protein (M2e) was engineered into the internal motif 5 of CMV CP and the recombinant gene then was transiently expressed in plants through a PVX vector. Chimeric CMV capsids reacted with specific antibodies produced to synthetic M2e epitope of the H5N1 strain of the virus. In addition, CMV CP-M2e protein was expressed to high levels in Escherichia coli bacterial cells and was recognized by antibodies to both CMV and M2e. This initial study demonstrates the feasibility of using plant virus-based vectors for expression of antigenic epitopes of H5N1 avian influenza in plants.

Orally Delivered, Plant-Produced Tat Protein Primes Mice For a Challenge DNA Vaccine Expressing Tat

The Tat protein has been recently explored as a prospective vaccine candidate against HIV-1 with broad, subtype non-specific action. A truncated version of Tat()Tat) with the basic loop, involved in immunosuppression, removed has been previously demonstrated as efficacious as the full-size Tat protein. We produced both full-size Tat and truncated _Tat in plants, including one edible species – spinach, thus simultaneously addressing problems of an inexpensive Tat production and a direct delivery through the mucosal route. We tested this oral delivery route in a mouse model. Tat and )Tat genes were assembled from a set of synthetic overlapping oligonucleotides, and subsequently cloned into a plant virus-based expression vector. Codon optimization allows production of up to 300–500 mg of Tat or )Tat antigen per 1 g of leaf tissue in spinach. Spinach plants inoculated with the Tat-producing constructs were collected and fed to mice 7–14 days post inoculation with or without mucosal adjuvants. Mice were fed with the Tat-producing or control vector-inoculated spinach. After 3 voluntary feedings, 1 week apart, 1 g per mice, no differences were detected in the growth rate or behavior of the animals fed with these two types of spinach. None of the animals developed measurable Tat antibodies. Challenge DNA vaccination with a homologous Tatexpressing construct was performed using a gene gun. Following DNA vaccination, however, mice previously receiving oral Tat with cholera toxin as an adjuvant, developed higher antibody titers to Tat than did the controls, with the titers peaking at four weeks post-vaccination. Thus, our data suggested that oral Tat primed for the development of Tat antibodies when mice were challengevaccinated with plasmid DNA for expression of Tat. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005