Targeted mutagenesis in Nicotiana tabacum ADF gene using shockwave-mediated ribonucleoprotein delivery increases osmotic stress tolerance.

DNA-free genome editing involves the direct introduction of ribonucleoprotein (RNP) complexes into cells, but this strategy has rarely been successful in plants. In the present study, we describe a new technique for the introduction of RNPs into plant cells involving the generation of cavitation bubbles using a pulsed laser. The resulting shockwave achieves the efficient transfection of walled cells in tissue explants by creating transient membrane pores. RNP-containing cells were rapidly identified by fluorescence microscopy, followed by regeneration and the screening of mutant plants by high-resolution melt analysis. We used this technique in Nicotiana tabacum to target the endogenous phytoene desaturase (PDS) and actin depolymerizing factor (ADF) genes. Genome-edited plants were produced with an efficiency of 35.2% for PDS and 16.5% for ADF. Further we evaluated the physiological, cellular and molecular effects of ADF mutations in T2 mutant plants under drought and salinity stress. The results suggest that ADF acts as a key regulator of osmotic stress tolerance in plants.

Biodistribution of Filamentous Plant Virus Nanoparticles: Pepino Mosaic Virus versus Potato Virus X.

Nanoparticles with high aspect ratios have favorable attributes for drug delivery and bioimaging applications based on their enhanced tissue penetration and tumor homing properties. Here, we investigated a novel filamentous viral nanoparticle (VNP) based on the Pepino mosaic virus (PepMV), a relative of the established platform Potato virus X (PVX). We studied the chemical reactivity of PepMV, produced fluorescent versions of PepMV and PVX, and then evaluated their biodistribution in mouse tumor models. We found that PepMV can be conjugated to various small chemical modifiers including fluorescent probes via the amine groups of surface-exposed lysine residues, yielding VNPs carrying payloads of up to 1600 modifiers per particle. Although PepMV and PVX share similarities in particle size and shape, PepMV achieved enhanced tumor homing and less nonspecific tissue distribution compared to PVX in mouse models of triple negative breast cancer and ovarian cancer. In conclusion, PepMV provides a novel tool for nanomedical research but more research is needed to fully exploit the potential of plant VNPs for health applications.

Combination of Plant Virus Nanoparticle-Based in Situ Vaccination with Chemotherapy Potentiates Antitumor Response.

Immunotherapeutics are gaining more traction in the armamentarium used to combat cancer. Specifically, in situ vaccination strategies have gained interest because of their ability to alter the tumor microenvironment to an antitumor state. Herein, we investigate whether flexuous plant virus-based nanoparticles formed by the potato virus X (PVX) can be used as an immunotherapeutic for in situ vaccine monotherapy. We further developed dual chemo-immunotherapeutics by incorporating doxorubicin (DOX) into PVX yielding a dual-functional nanoparticle (PVX-DOX) or by coadministration of the two therapeutic regimes, PVX immunotherapy and DOX chemotherapy (PVX+DOX). In the context of B16F10 melanoma, PVX was able to elicit delayed tumor progression when administered as an intratumoral in situ vaccine. Furthermore, the coadministration of DOX via PVX+DOX enhanced the response of the PVX monotherapy through increased survival, which was also represented in the enhanced antitumor cytokine/chemokine profile stimulated by PVX+DOX when compared to PVX or DOX alone. Importantly, coadministered PVX+DOX was better for in situ vaccination than PVX loaded with DOX (PVX-DOX). Whereas the nanomedicine field strives to design multifunctional nanoparticles that integrate several functions and therapeutic regimens into a single nanoparticle, our data suggest a paradigm shift; some therapeutics may need to be administered separately to synergize and achieve the most potent therapeutic outcome. Altogether, our studies show that development of plant viral nanoparticles for in situ vaccines for treatment is a possibility, and dual mechanistic therapeutics can increase efficacy. Nonetheless, combining immunotherapeutics with cytolytic chemotherapy requires detailed investigation to inform optimal integration of cytolytic and immunotherapies and maximize synergy and efficacy.

Chemical addressability of potato virus X for its applications in bio/nanotechnology.

Potato virus X (PVX), a type member of the plant virus potexvirus group, offers a unique nanotechnology platform based on its high aspect ratio and flexible filamentous shape. The PVX platform has already been engineered and studied for its uses in imaging, drug delivery, and immunotherapies. While genetic engineering procedures are well established for PVX, there is limited information about chemical conjugation strategies for functionalizing PVX, partly due to the lack of structural information of PVX at high resolution. To overcome these challenges, we built a structural model of the PVX particle based on the available structures from pepino mosaic virus (PepMV), a close cousin of PVX. Using the model and a series of chemical conjugation experiments, we identified and probed the addressability of cysteine side chains. Chemical reactivity of cysteines was confirmed using Michael-addition and thiol-selective probes, including fluorescent dyes and biotin tags. LC/MS/MS was used to map Cys 121 as having the highest selectivity for modification. Finally, building on the availability of two reactive groups, the newly identified Cys and previously established Lys side chains, we prepared multifunctional PVX nanoparticles by conjugating Gd-DOTA for magnetic resonance imaging (MRI) to lysines and fluorescent dyes for optical imaging to cysteines. The resulting functionalized nanofilament could have applications in dual-modal optical-MRI imaging applications. These results further extend the understanding of the chemical properties of PVX and enable development of novel multifunctional platforms in bio/nanotechnology.

Engineering Potato Virus X Particles for a Covalent Protein Based Attachment of Enzymes.

Plant virus nanoparticles are often used to display functional amino acids or small peptides, thus serving as building blocks in application areas as diverse as nanoelectronics, bioimaging, vaccination, drug delivery, and bone differentiation. This is most easily achieved by expressing coat protein fusions, but the assembly of the corresponding virus particles can be hampered by factors such as the fusion protein size, amino acid composition, and post-translational modifications. Size constraints can be overcome by using the Foot and mouth disease virus 2A sequence, but the compositional limitations cannot be avoided without the introduction of time-consuming chemical modifications. SpyTag/SpyCatcher technology is used in the present study to covalently attach the Trichoderma reesei endoglucanase Cel12A to Potato virus X (PVX) nanoparticles. The formation of PVX particles is confirmed by western blot, and the ability of the particles to display Cel12A is demonstrated by enzyme-linked immunosorbent assays and transmission electron microscopy. Enzymatic assays show optimal reaction conditions of 50 °C and pH 6.5, and an increased substrate conversion rate compared to free enzymes. It is concluded that PVX displaying the SpyTag can serve as new scaffold for protein display, most notably for proteins with post-translational modifications.

Cell wall modification in tobacco by differential targeting of recombinant endoglucanase from Trichoderma reesei.

BACKGROUND: The development of transgenic plants as a production platform for biomass-degrading enzymes is a promising tool for an economically feasible allocation of enzymes processing lignocellulose. Previous research has already identified a major limitation of in planta production such as interference with the structure and integrity of the plant cell wall resulting in a negative influence on plant growth and development. RESULTS: Here, we describe the in planta expression of endoglucanase TrCel5A from the mesophilic fungus Trichoderma reesei with differential intracellular targeting and evaluate its impact on the tobacco cell wall composition. Targeting of the enzyme to the apoplast leads to distinct changes in cell polysaccharides such as glucose level in the matrix polysaccharides (MPS). These effects are combined with severe changes in plant development. Retention of TrCel5A in the endoplasmic reticulum (ER) could avoid visible effects on plant growth under the chosen conditions, but exhibits changes in the composition of the MPS. CONCLUSIONS: These results give new insights into the complex interaction of heterologous cellulase expression with cell wall development and it outlines novel promising strategies to engineer plant cell walls for improved biomass processing.

Simple and portable magnetic immunoassay for rapid detection and sensitive quantification of plant viruses.

Plant pathogens cause major economic losses in the agricultural industry because late detection delays the implementation of measures that can prevent their dissemination. Sensitive and robust procedures for the rapid detection of plant pathogens are therefore required to reduce yield losses and the use of expensive, environmentally damaging chemicals. Here we describe a simple and portable system for the rapid detection of viral pathogens in infected plants based on immunofiltration, subsequent magnetic detection, and the quantification of magnetically labeled virus particles. Grapevine fanleaf virus (GFLV) was chosen as a model pathogen. Monoclonal antibodies recognizing the GFLV capsid protein were immobilized onto immunofiltration columns, and the same antibodies were linked to magnetic nanoparticles. GFLV was quantified by immunofiltration with magnetic labeling in a double-antibody sandwich configuration. A magnetic frequency mixing technique, in which a two-frequency magnetic excitation field was used to induce a sum frequency signal in the resonant detection coil, corresponding to the virus concentration within the immunofiltration column, was used for high-sensitivity quantification. We were able to measure GFLV concentrations in the range of 6 ng/ml to 20 µg/ml in less than 30 min. The magnetic immunoassay could also be adapted to detect other plant viruses, including Potato virus X and Tobacco mosaic virus, with detection limits of 2 to 60 ng/ml.

Metabolic engineering for p-coumaryl alcohol production in Escherichia coli by introducing an artificial phenylpropanoid pathway.

The plant polymer lignin is the greatest source of aromatic chemical structures on earth. Hence, the chemically diverse lignin monomers are valuable raw materials for fine chemicals, materials synthesis, and food and flavor industries. However, extensive use of this natural resource is hampered by the large number of different lignin monomers and the complex and irregular structure of lignin, which renders current processes for its chemical or enzymatic degradation inefficient. The microbial production of lignin monomers from renewable resources represents a promising alternative to lignin degradation, which could meet the demand for aromatic chemical structures. In this study, we describe the functional introduction of an artificial phenylpropanoid pathway into Escherichia coli, achieved by transferring several genes from plants and microbes. The established chimeric pathway efficiently converts l-tyrosine into the lignin precursor molecule p-coumaryl alcohol.

A Combination of Flexible Modified Plant Virus Nanoparticles Enables Additive Effects Resulting in Improved Osteogenesis.

Plant virus nanoparticles (VNPs) genetically engineered to present osteogenic cues provide a promising method for biofunctionalizing hydrogels in bone tissue engineering. Flexible Potato virus X (PVX) nanoparticles substantially enhance the attachment and differentiation of human mesenchymal stem cells (hMSCs) by presenting the RGD motif, hydroxyapatite-binding peptide (HABP), or consecutive polyglutamates (E8) in a concentration-dependent manner. Therefore, it is hypothesized that Tobacco mosaic virus nanoparticles, which present 1.6 times more functional peptides than PVX, will meliorate such an impact. This study hypothesizes that cultivating hMSCs on a surface coated with a combination of two VNPs presenting peptides for either cell attachment or mineralization can achieve additionally enhancing effects on osteogenesis. Calcium minerals deposited by differentiating hMSCs increases two to threefold for this combination, while the Alkaline Phosphatase activity of hMSCs grown on the PVX-RGD/PVX-HABP-coated surface significantly surpasses any other VNP combination. Superior additive effects are observed for the first time by employing a combination of VNPs with varying functionalities. It is found that the flexible VNP geometry plays a more critical role than the concentration of functional peptides. In conclusion, various peptide-presenting plant VNPs exhibit an additive enhancing effect offering significant potential for effectively functionalizing cell-containing hydrogels in bone tissue engineering.

Flow cytometry-based ultra-high-throughput screening assay for cellulase activity.

We have developed a novel, ultra-high-throughput screening assay for the detection of cellulase activity based on fluorescence-activated cell sorting (FACS) and double emulsion technology. Cellulase activity is detected using a series of coupled enzymes, including hexose oxidase (HOx), which generates hydrogen peroxide from the reducing sugars released by cellulases in the presence of any natural or artificial substrate. The assay can be adapted to suit a microtiter plate format, but the highest throughput is achieved by using FACS to screen high-complexity cellulase clone libraries. Using this approach, we achieved a 12-fold enrichment of positive (cellulase-expressing) cells in cellulase reference libraries after just one sorting round.

In vivo expression and binding activity of scFv-RWAV, which recognizes the coat protein of tomato leaf curl New Delhi virus (family Geminiviridae).

Recombinant antibodies expressed in plants have the potential to interrupt virus infections by blocking essential stages of the infection cycle. Here, we show that the expression of a recombinant single-chain variable fragment (scFv) that recognizes the coat protein of tomato leaf curl New Delhi virus (ToLCNDV) in vitro can also bind to a recombinant coat protein in vivo in the reducing environment of the plant cytosol. The scFv and its target were both expressed as fluorescent protein fusions, one incorporating green fluorescent protein (GFP) and the other DsRed. We found that the incorporation of a nuclear localization signal into the scFv construct resulted in the nuclear import of the antibody-antigen complex, as shown by colocalization of the two fluorescent signals. This demonstrates that recombinant antibodies can be targeted to the nucleus and will bind to geminivirus coat proteins therein, allowing the virus infection cycle to be interrupted during its critical replicative phase.

Advanced Fusion Strategies for the Production of Functionalized Potato Virus X Virions.

Plant virions are ideal for nanotechnology applications because they are structurally diverse and can self-assemble naturally, allowing for large-scale production in plants by molecular farming. Potato virus X (PVX) is particularly amenable due to the unique properties of its filamentous and flexible capsid, but efficient strategies are required to adapt the surface properties of PVX, such as the attachment of proteins and peptides. This chapter describes the selection and utilization of 2A ribosomal skip sequences, allowing the presentation of heterologous proteins and peptides as N-terminal fusions to the PVX coat protein at different densities. Another strategy for the rapid modification of PVX capsids is the plug-and-display module of the SpyTag/SpyCatcher system. The SpyTag can be presented on the PVX surface, allowing for the attachment of any protein fused to the SpyCatcher sequence.

Plug-and-Display Photo-Switchable Systems on Plant Virus Nanoparticles.

Light can be used to regulate protein interactions with a high degree of spatial and temporal precision. Photo-switchable systems therefore allow the development of controllable protein complexes that can influence various cellular and molecular processes. Here, we describe a plant virus-based nanoparticle shuttle for the distribution of proteins that can be released when exposed to light. Potato virus X (PVX) is often used as a presentation system for heterologous proteins and epitopes, and has ideal properties for biomedical applications such as good tissue penetration and the ability to form hydrogels that present signaling molecules and promote cell adhesion. In this study, we describe three different systems attached to the surface of PVX particles: LOVTRAP, BphP1/QPAS1 and Dronpa145N. We demonstrated the functionality of all three photo-switchable protein complexes in vitro and the successful loading and unloading of PVX particles. The new systems provide the basis for promising applications in the biomedical and biomaterial sciences.

A targeted metabolomics method for extra- and intracellular metabolite quantification covering the complete monolignol and lignan synthesis pathway.

Microbial synthesis of monolignols and lignans from simple substrates is a promising alternative to plant extraction. Bottlenecks and byproduct formation during heterologous production require targeted metabolomics tools for pathway optimization. In contrast to available fractional methods, we established a comprehensive targeted metabolomics method. It enables the quantification of 17 extra- and intracellular metabolites of the monolignol and lignan pathway, ranging from amino acids to pluviatolide. Several cell disruption methods were compared. Hot water extraction was best suited regarding monolignol and lignan stability as well as extraction efficacy. The method was applied to compare enzymes for alleviating bottlenecks during heterologous monolignol and lignan production in E. coli. Variants of tyrosine ammonia-lyase had a considerable influence on titers of subsequent metabolites. The choice of multicopper oxidase greatly affected the accumulation of lignans. Metabolite titers were monitored during batch fermentation of either monolignol or lignan-producing recombinant E. coli strains, demonstrating the dynamic accumulation of metabolites. The new method enables efficient time-resolved targeted metabolomics of monolignol- and lignan-producing E. coli. It facilitates bottleneck identification and byproduct quantification, making it a valuable tool for further pathway engineering studies. This method will benefit the bioprocess development of biotransformation or fermentation approaches for microbial lignan production.

Potato virus X as a novel platform for potential biomedical applications.

We demonstrate that nanoparticles formed from the rod-shaped plant virus Potato virus X (PVX) can serve as a novel platform for biomedical applications. Bioconjugation protocols including amine modification and "click" chemistry allowed the efficient functionalization of PVX with biotins, dyes, and PEGs. Fluorescent-labeled and PEGylated PVX particles revealed that different fluorescent labels have a profound effect on PVX-cell interactions. Applying bioconjugation chemistries to PVX opens the door for chemical functionalization with targeting and therapeutic molecules.

Analysis of Engineered Tobacco Mosaic Virus and Potato Virus X Nanoparticles as Carriers for Biocatalysts.

Plant virus nanoparticles are promising candidates for the development of novel materials, including nanocomposites and scaffolds/carriers for functional molecules such as enzymes. Their advantages for enzyme immobilization include a modular organization, a robust and programmable structure, and a simple, cost-effective production. However, the activity of many enzymes relies on posttranslational modification and most plant viruses replicate in the cytoplasm, so functional enzymes cannot be displayed on the virus surface by direct coat protein fusions. An alternative display system to present the Trichoderma reesei endoglucanase Cel12A on potato virus X (PVX) using SpyTag/SpyCatcher (ST/SC) technology was recently developed by the authors, which allows the carrier and enzyme to be produced separately before isopeptide conjugation. Although kinetic analysis clearly indicated efficient biocatalyst activity, the PVX carrier interfered with substrate binding. To overcome this, the suitability of tobacco mosaic virus (TMV) was tested, which can also accommodate a larger number of ST peptides. We produced TMV particles displaying ST as a new platform for the immobilization of enzymes such as Cel12A, and compared its performance to the established PVX-ST platform in terms of catalytic efficiency. Although more enzyme molecules were immobilized on the TMV-ST particles, we found that the rigid scaffold and helical spacing significantly affected enzyme activity.

Three Alternative Treatment Protocols for the Efficient Inactivation of Potato Virus X.

Filamentous nanomaterials are flexible with a high aspect ratio, conferring unique mechanical, electromagnetic, and optical properties; promoting tissue penetration; and allowing the formation of hierarchical superstructures. The fabrication of synthetic nanofilaments with uniform properties is challenging, but this can be addressed by the use of filamentous plant viruses such as potato virus X (PVX), which are produced as monodisperse structures from a genetic template. To take advantage of PVX without risks to agriculture and the environment, it is necessary to inactivate the virus efficiently without disrupting its chemical and material properties. Herein, we report experiments showing that PVX can be completely inactivated by exposure to UV irradiation (0.5 J cm-2) or chemical treatment (1 mM ß-propiolactone or 10 mM formalin) without interfering with the chemical addressability of lysine or cysteine residues, which are typically used as conjugation handles for virus nanoparticle functionalization.

From infection to healing: The use of plant viruses in bioactive hydrogels.

Plant viruses show great diversity in shape and size, but each species forms unique nucleoprotein particles that are symmetrical and monodisperse. The genetically programed structure of plant viruses allows them to be modified by genetic engineering, bioconjugation, or encapsulation to form virus nanoparticles (VNPs) that are suitable for a broad range of applications. Plant VNPs can be used to present foreign proteins or epitopes, to construct inorganic hybrid materials, or to carry molecular cargos, allowing their utilization as imaging reagents, immunomodulators, therapeutics, nanoreactors, and biosensors. The medical applications of plant viruses benefit from their inability to infect and replicate in human cells. The structural properties of plant viruses also make them useful as components of hydrogels for tissue engineering. Hydrogels are three-dimensional networks composed of hydrophilic polymers that can absorb large amounts of water. They are used as supports for tissue regeneration, as reservoirs for controlled drug release, and are found in contact lenses, many wound healing materials, and hygiene products. They are also useful in ecological applications such as wastewater treatment. Hydrogel-based matrices are structurally similar to the native extracellular matrix (ECM) and provide a scaffold for the attachment of cells. To fully replicate the functions of the ECM it is necessary to augment hydrogels with biological cues that regulate cellular interactions. This can be achieved by incorporating functionalized VNPs displaying ligands that influence the mechanical characteristics of hydrogels and their biological properties, promoting the survival, proliferation, migration, and differentiation of embedded cells. This article is categorized under: Implantable Materials and Surgical Technologies > Nanomaterials and Implants Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement.

Generation and characterization of a scFv against recombinant coat protein of the geminivirus tomato leaf curl New Delhi virus.

We report the establishment of a hybridoma cell line secreting the monoclonal antibody (mAb) HAV, which recognizes the coat (AV1) protein of tomato leaf curl New Delhi virus (ToLCNDV), a begomovirus. The cell line was obtained following immunization of mice with purified recombinant AV1 fused to glutathione S-transferase (GST). A single-chain variable fragment (scFv-SAV) was assembled from hybridoma cDNA, but sequence analysis revealed a single nucleotide deletion causing a frame shift that resulted in a 21-residue N-terminal truncation. The missing nucleotide was restored by in vitro site-directed mutagenesis to create scFv-RWAV. The binding properties of mAb HAV and the corresponding scFvs were characterized by western blot, ELISA and surface plasmon resonance spectroscopy. MAb HAV bound to AV1 with nanomolar affinity but reacted neither with the N-terminal region of the protein nor with the GST fusion partner. This suggested that the antibody recognized a linear epitope in a region of the coat protein that is conserved among begomoviruses. Both scFvs retained the antigen specificity of mAb HAV, although the dissociation rate constant of scFv-RWAV was tenfold greater than that of scFv-SAV, showing the importance of restoring the 21 N-terminal amino acids.

Attachment of Ultralow Amount of Engineered Plant Viral Nanoparticles to Mesenchymal Stem Cells Enhances Osteogenesis and Mineralization.

Hydrogel-based materials are widely used to mimic the extracellular matrix in bone tissue engineering, although they often lack biofunctional cues. In the authors' previous work, Potato virus X (PVX), a flexible rod-shaped biocompatible plant virus nanoparticle (VNP) with 1270 coat protein subunits, is genetically modified to present functional peptides for generating a bone substitute. Here, PVX is engineered to present mineralization- and osteogenesis-associated peptides and laden in hydrogels at a concentration lower by two orders of magnitude. Its competence in mineralization is demonstrated both on 2D surfaces and in hydrogels and the superiority of enriched peptides on VNPs is verified and compared with free peptides and VNPs presenting fewer functional peptides. Alkaline phosphatase activity and Alizarin red staining of human mesenchymal stem cells increase 1.2-1.7 times when stimulate by VNPs. Engineered PVX adheres to cells, exhibiting a stimulation of biomimetic peptides in close proximity to the cells. The retention of VNPs in hydrogels is monitored and more than 80% of VNPs remain inside after several washing steps. The mechanical properties of VNP-laden hydrogels are investigated, including viscosity, gelling temperature, and compressive tangent modulus. This study demonstrates that recombinant PVX nanoparticles are excellent candidates for hydrogel nanocomposites in bone tissue engineering.