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.
Potato virus X (PVX), a species of the genus Potexvirus, is a plant pathogenic virus that causes severe symptoms such as mild mosaic, crinkling, necrosis, and mottling on leaves. The objectives of ...the present study were to investigate the effect of PVX virus infection on the metabolic system in nontransgenic and Arabidopsis thaliana production of anthocyanin pigment 1 (AtPAP1) transgenic tobacco using transcript expression analysis and metabolic profiling. Potato virus X inoculation increased the gene expression of phenylpropanoid and flavonoid biosynthesis and the production of chlorogenic acid, p-coumaric acid, benzoic acid, rutin, quercetin, and kaempferol in nontransgenic tobacco leaves. However, in the AtPAP1 transgenic tobacco leaves, PVX inoculation decreased the expression of AtPAP1 and phenylpropanoid and flavonoid biosynthesis genes, and the production of phenolics and anthocyanin also declined. In contrast, the levels of amino acids and tricarboxylic acid (TCA) cycle intermediates increased after infection in the AtPAP1 transgenic plant leaves. To date, these results have not been reported previously. We suggest that PVX infection decreases AtPAP1 expression, leading to the downregulation of phenylpropanoid and flavonoid biosynthesis in transgenic plants.
We propose nucleic acid lateral flow assay (LFA) coupled with reverse transcription recombinase polymerase amplification (RT-RPA) resulting from step-by-step multiparametric adjustments to both ...RT-RPA reactions and LFA interactions. The assay was realized for RNA virus detection using the example of potato virus X (PVX), a dangerous phytopathogen. The assay stages were adjusted for sensitive detection. (1) DNA target was designed and verified. A fragment (146 bp) of coat protein gene (gp5) and biotin-/fluorescein-labeled forward/reverse primers were chosen to produce target amplicons. (2) In a test strip, the construction advantage of the realization of the highest affinity interaction (biotin–streptavidin in our research) through gold nanoparticle conjugate (streptavidin immobilized on the GNP surface) was demonstrated. (3) RPA with reverse transcription was adjusted including primer concentration, order of components’ mixing, and reaction temperature. Due to the adjustments, the assay was able to detect 0.14 ng PVX per g potato leaves at 30 min. The PVX assay was 260 times more sensitive than conventional lateral flow assay based on antibodies and demonstrated the same sensitivity as PCR detection. The proposed adjustments are applicable for ultrasensitive and rapid detection of various RNA viruses.
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•Recombinase polymerase amplification – lateral flow assay for PVX was developed.•Detection limit was 0.14 ng potato virus X (PVX) per g potato leaves.•Sensitivity was 260-fold increased as compared with antibody-based lateral flow assay.•Multiparametric adjustments for detection of RNA virus were proposed.
The potato (Solanum tuberosum) nucleotide binding-leucine-rich repeat immune receptor Rx confers resistance to Potato virus X (PVX) and requires Ran GTPase-activating protein 2 (RanGAP2) for ...effective immune signaling. Although Rx does not contain a discernible nuclear localization signal, the protein localizes to both the cytoplasm and nucleus in Nicotiana benthamiana. Transient coexpression of Rx and cytoplasmically localized RanGAP2 sequesters Rx in the cytoplasm. This relocation of the immune receptor appeared to be mediated by the physical interaction between Rx and RanGAP2 and was independent of the concomitant increased GAP activity. Coexpression with RanGAP2 also potentiates Rx-mediated immune signaling, leading to a hypersensitive response (HR) and enhanced resistance to PVX. Besides sequestration, RanGAP2 also stabilizes Rx, a process that likely contributes to enhanced defense signaling. Strikingly, coexpression of Rx with the Rx-interacting WPP domain of RanGAP2 fused to a nuclear localization signal leads to hyperaccumulation of both the WPP domain and Rx in the nucleus. As a consequence, both Rx-mediated resistance to PVX and the HR induced by auto-active Rx mutants are significantly suppressed. These data show that a balanced nucleocytoplasmic partitioning of Rx is required for proper regulation of defense signaling. Furthermore, our data indicate that RanGAP2 regulates this partitioning by serving as a cytoplasmic retention factor for Rx.
•AGO2-deficient plants infected with potato virus X show systemic necrosis.•Systemic necrosis was associated with reduction of the CCS1 mRNA level.•Genetic proof implicates CCS1 in systemic necrosis.
...RNA silencing is a prominent antiviral defense mechanism in plants. When infected with a virus, RNA silencing–deficient plants tend to show exacerbated symptoms along with increased virus accumulation. However, how symptoms are exacerbated is little understood. Here, we investigated the role of the copper chaperon for superoxide dismutase (CCS) 1, in systemic necrosis observed in Argonaute (AGO)2-silenced tomato plants infected with potato virus X (PVX). While infection with the UK3 strain of PVX induced mosaic symptoms in tomato plants, systemic necrosis occurred when AGO2 was silenced. The CCS1 mRNA level was reduced and micro RNA398 (miR398), which potentially target CCS1, was increased in AGO2-knockdown tomato plants infected with PVX-UK3. Ectopic expression of CCS1 using recombinant PVX attenuated necrosis, suggesting that CCS1 alleviates systemic necrosis by activating superoxide dismutases to scavenge reactive oxygen species. Previous reports have indicated a decrease in the levels of CCS1 and superoxide dismutases along with an increased level of miR398 in plants infected with other viruses and viroids, and thus might represent shared regulatory mechanisms that exacerbate symptoms in these plants.
Abstract
The chloroplast protein ferredoxin 1 (FD1), with roles in the chloroplast electron transport chain, is known to interact with the coat proteins (CPs) of Tomato mosaic virus and Cucumber ...mosaic virus. However, our understanding of the roles of FD1 in virus infection remains limited. Here, we report that the Potato virus X (PVX) p25 protein interacts with FD1, whose mRNA and protein levels are reduced by PVX infection or by transient expression of p25. Silencing of FD1 by Tobacco rattle virus-based virus-induced gene silencing (VIGS) promoted the local and systemic infection of plants by PVX. Use of a drop-and-see (DANS) assay and callose staining revealed that the permeability of plasmodesmata (PDs) was increased in FD1-silenced plants together with a consistently reduced level of PD callose deposition. After FD1 silencing, quantitative reverse transcription–real-time PCR (qRT–PCR) analysis and LC-MS revealed these plants to have a low accumulation of the phytohormones abscisic acid (ABA) and salicylic acid (SA), which contributed to the decreased callose deposition at PDs. Overexpression of FD1 in transgenic plants manifested resistance to PVX infection, but the contents of ABA and SA, and the PD callose deposition were not increased in transgenic plants. Overexpression of FD1 interfered with the RNA silencing suppressor function of p25. These results demonstrate that interfering with FD1 function causes abnormal plant hormone-mediated antiviral processes and thus enhances PVX infection.
Interference with ferrodoxin 1 function results in low accumulation of callose at plasmodesmata and abnormal plant hormone-mediated antiviral processes, thus facilitating PVX infection.
This study presents the joint use of magnetic nanoparticles (MNPs) and gold nanoparticles (GNPs) for double enhancement in a lateral flow immunoassay (LFIA). The study realizes two types of ...enhancement: (1) increasing the concentration of analytes in the samples using conjugates of MNPs with specific antibodies and (2) increasing the visibility of the label through MNP aggregation caused by GNPs. The proposed strategy was implemented using a LFIA for potato virus X (PVX), a significant potato pathogen. MNPs conjugated with biotinylated antibodies specific to PVX and GNPs conjugated with streptavidin were synthesized and characterized. The LFIAs with and without the proposed enhancements were compared. The double-enhanced LFIA achieved the highest sensitivity, equal to 0.25 ng mL−1 and 32 times more sensitivity than the non-enhanced LFIA (detection limit: 8 ng mL−1). LFIAs using one of the types of amplification (magnetic concentration without GNPs-causing aggregation or MNP aggregation without the concentration stage) showed intermediate levels of sensitivity. The double-enhanced LFIA was successfully used for PVX detection in potato leaves. The results for PVX detection in the infected plants were similar for the double-enhanced LFIA developed and the conventional LFIA based on the GNP conjugates; however, the new system provided significant coloring enhancement. This study confirmed that a simple combination of MNPs and GNPs has great potential for high-sensitivity detection and could possibly be adopted for LFIAs of other compounds.
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•Magnetic and gold nanoparticles (MNPs & GNPs) were used together in lateral flow immunoassay.•Heteroaggregates of MNPs and GNPs increased the assay sensitivity.•Combination of magnetic concentration and MNP & GNP aggregates had a cumulative effect.•The double enhancement increased the sensitivity of potato virus Х detection by 32-fold.
SUMMARY
Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR‐associated proteins (Cas) have revolutionized genome editing in many organisms, including ...plants. Most CRISPR‐Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target‐specific and a screening process is usually required to identify those most effective. Plant virus‐derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus‐induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX‐based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus‐free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR‐Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops.
Significance Statement
Plant virus‐derived vectors allow fast and efficient delivery of synthetic guide RNAs into adult plants for CRISPR‐Cas‐based genome editing. We engineered a Potato virus X vector for CRISPR‐Cas genome editing of solanaceous plants. This vector expresses unspaced arrays of synthetic guide RNAs and achieves multiplex editing in adult plant tissues in a few days. Virus‐free multiplex genome‐edited plants with biallelic mutations can be easily obtained from inoculated plants.
Plant viral nanoparticles (VNPs) are a novel class of nanocarriers with implications for drug delivery in cancer therapy. VNPs are characterized by their highly symmetrical nanoscale structures. ...Furthermore, plant VNPs are biocompatible, biodegradable, and non-infectious in mammals. VNPs provide a proteinaceous platform technology that can be readily engineered to carry contrast agents and therapies using chemical and genetic modifications. Of particular interest are high aspect ratio, elongated filaments such as the ones formed by potato virus X (PVX, measuring 515 × 13 nm). PVX has demonstrated enhanced tumor homing and penetration properties compared to spherical counterparts. Here, we sought to investigate the potential of PVX as a drug carrier delivering doxorubicin (DOX), a commonly used cancer chemotherapy. We synthesized therapeutic PVX nanoparticles using a simple in-solution mixing protocol; after 5 days of mixing of DOX and PVX and ultra-centrifugal purification, ∼1000 DOX per PVX were stably associated with the carrier, most likely based on hydrophobic interaction. Efficacy and drug activity of PVX-DOX were confirmed using a panel of cancer cell lines including ovarian cancer, breast cancer, and cervical cancer. Lastly, we demonstrated treatment of athymic mice bearing human MDA-MB-231 breast cancer xenografts: PVX-DOX treatment resulted in reduced tumor growth in this model. Our results open the door for further development of PVX and other high aspect ratio plant VNPs for applications in cancer therapy.
Plant viral nanoparticles (VNPs) are a novel class of nanocarriers with implications for drug delivery in cancer therapy.
Infections of plants by multiple viruses are common in nature and may result in synergisms in pathologies. Several environmental factors influence plant-virus interactions and act on virulence and ...host defense responses. Mixed viral infections may be more frequent under environmental conditions associated with global warming. Here, we address how changes in the two main parameters behind global warming, carbon dioxide concentrations (CO sub(2)) and temperature, may affect virulence of Potato virus X(PVX)/potyvirus-associated synergism compared with single infections in Nicotiana benthamiana. Elevated CO sub(2) resulted in attenuated virulence of single infection by PVX, which correlated with a lower accumulation of virus. In contrast, virulence of PVX/potyvirus-associated synergism was maintained at elevated CO sub(2). On the other hand, elevated temperature decreased markedly both virulence and virus titers in the synergistic infection. We also show that the HR-like response elicited by transient coexpression of PVX P25 together with the potyviral helper component-proteinase protein was significantly enhanced by elevated temperature, whereas it was reduced by elevated CO sub(2). Both proteins are main pathogenicity determinants in PVX-associated synergisms. These findings indicate that, under environmental conditions associated with global warming, virulence of PVX/potyvirus-associated synergisms is expected to vary relative to single infections and, thus, may have pathological consequences in the future.
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