Systemic Infection of Nicotiana benthamiana with Potato virus X Nanoparticles Presenting a Fluorescent iLOV Polypeptide Fused Directly to the Coat Protein

Plant virus-based nanoparticles can be produced in plants on a large scale and are easily modified to introduce new functions, making them suitable for applications such as vaccination and drug delivery, tissue engineering, and in vivo imaging. The latter is often achieved using green fluorescent protein and its derivatives, but the monovalent fluorescent protein iLOV is smaller and more robust. Here, we fused the iLOV polypeptide to the N-terminus of the Potato virus X (PVX) coat protein, directly or via the Foot-and-mouth disease virus 2A sequence, for expression in Nicotiana benthamiana. Direct fusion of the iLOV polypeptide did not prevent the assembly or systemic spread of the virus and we verified the presence of fusion proteins and iLOV hybrid virus particles in leaf extracts. Compared to wild-type PVX virions, the PVX particles displaying the iLOV peptide showed an atypical, intertwined morphology. Our results confirm that a direct fusion of the iLOV fluorescent protein to filamentous PVX nanoparticles offers a promising tool for imaging applications.

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Corrigendum to “Nicotiana benthamiana protein, NPCIP1, interacting with Potato virus X coat protein plays a role as susceptible factor for viral infection” [Virology 386 (2009) 257–269]

Virology ◽

10.1016/j.virol.2009.06.019 ◽

2009 ◽

Vol 391 (1) ◽

pp. 149-150

Author(s):

Mi-Ri Park ◽

Sang-Ho Park ◽

Sang-Yun Cho ◽

Kook-Hyung Kim

Keyword(s):

Coat Protein ◽

Viral Infection ◽

Potato Virus ◽

Nicotiana Benthamiana ◽

Potato Virus X

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Phenotypes and Functional Effects Caused by Various Viral RNA Silencing Suppressors in Transgenic Nicotiana benthamiana and N. tabacum

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-2-0178 ◽

2008 ◽

Vol 21 (2) ◽

pp. 178-187 ◽

Cited By ~ 50

Author(s):

Shahid Aslam Siddiqui ◽

Cecilia Sarmiento ◽

Erkki Truve ◽

Harry Lehto ◽

Kirsi Lehto

Keyword(s):

Mosaic Virus ◽

Potato Virus ◽

Nicotiana Benthamiana ◽

Rna Silencing ◽

Fluorescent Protein ◽

Potato Virus X ◽

Mottle Virus ◽

African Cassava Mosaic Virus ◽

Rice Yellow Mottle Virus ◽

Green Fluorescent

RNA silencing suppressor genes derived from six virus genera were transformed into Nicotiana benthamiana and N. tabacum plants. These suppressors were P1 of Rice yellow mottle virus (RYMV), P1 of Cocksfoot mottle virus, P19 of Tomato bushy stunt virus, P25 of Potato virus X, HcPro of Potato virus Y (strain N), 2b of Cucumber mosaic virus (strain Kin), and AC2 of African cassava mosaic virus (ACMV). HcPro caused the most severe phenotypes in both Nicotiana spp. AC2 also produced severe effects in N. tabacum but a much milder phenotype in N. benthamiana, although both HcPro and AC2 affected the leaf tissues of the two Nicotiana spp. in similar ways, causing hyperplasia and hypoplasia, respectively. P1-RYMV caused high lethality in the N. benthamiana plants but only mild effects in the N. tabacum plants. Phenotypic alterations produced by the other transgenes were minor in both species. Interestingly, the suppressors had very different effects on crucifer-infecting Tobamovirus (crTMV) infections. AC2 enhanced both spread and brightness of the crTMV-green fluorescent protein (GFP) lesions, whereas 2b and both P1 suppressors enhanced spread but not brightness of these lesions. P19 promoted spread of the infection into new foci within the infiltrated leaf, whereas HcPro and P25 suppressed the spread of crTMV-GFP lesions.

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Expression of a recombinant Human papillomavirus 16 E6GT oncoprotein fused to N- and C-termini of Potato virus X coat protein in Nicotiana benthamiana

Plant Cell Tissue and Organ Culture (PCTOC) ◽

10.1007/s11240-012-0253-3 ◽

2012 ◽

Vol 113 (1) ◽

pp. 81-90 ◽

Cited By ~ 5

Author(s):

Noemi Cerovska ◽

Tomas Moravec ◽

Hana Hoffmeisterova ◽

Helena Plchova ◽

Helena Synkova ◽

...

Keyword(s):

Human Papillomavirus ◽

Coat Protein ◽

Potato Virus ◽

Nicotiana Benthamiana ◽

Potato Virus X ◽

Human Papillomavirus 16

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Evidence that the Linker between the Methyltransferase and Helicase Domains of Potato Virus X Replicase Is Involved in Homologous RNA Recombination

Journal of Virology ◽

10.1128/jvi.00179-08 ◽

2009 ◽

Vol 83 (15) ◽

pp. 7761-7769 ◽

Cited By ~ 7

Author(s):

Heidrun-Katharina Draghici ◽

Mark Varrelmann

Keyword(s):

Coat Protein ◽

Potato Virus ◽

Fluorescent Protein ◽

Triple Gene Block ◽

Leaf Tissue ◽

Potato Virus X ◽

Wild Type Virus ◽

Rna Recombination ◽

Gene Block

ABSTRACT Recombination in RNA viruses, one of the main factors contributing to their genetic variability and evolution, is a widespread phenomenon. In this study, an in vivo assay to characterize RNA recombination in potato virus X (PVX), under high selection pressure, was established. Agrobacterium tumefaciens was used to express in Nicotiana benthamiana leaf tissue both a PVX isolate labeled with green fluorescent protein (GFP) containing a coat protein deletion mutation (ΔCP) and a transcript encoding a functional coat protein +3′-ntr. Coexpression of the constructs led to virus movement and systemic infection; reconstituted recombinants were observed in 92% of inoculated plants. Similar results were obtained using particle bombardment, demonstrating that recombination mediated by A. tumefaciens was not responsible for the occurrence of PXC recombinants. The speed of recombination could be estimated by agroinfection of two PVX mutants lacking the 3′ and 5′ halves of the genome, respectively, with an overlap in the triple gene block 1 gene, allowing GFP expression only in the case of recombination. Ten different pentapeptide insertion scanning replicase mutants with replication abilities comparable to wild-type virus were applied in the different recombination assays. Two neighboring mutants affecting the linker between the methyltransferase and helicase domains were shown to be strongly debilitated in their ability to recombine. The possible functional separation of replication and recombination in the replicase molecule supports the model that RNA recombination represents a distinct function of this protein, although the underlying mechanism still needs to be investigated.

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Potato virus X movement in Nicotiana benthamiana: new details revealed by chimeric coat protein variants

Molecular Plant Pathology ◽

10.1111/j.1364-3703.2011.00739.x ◽

2011 ◽

Vol 13 (2) ◽

pp. 198-203 ◽

Cited By ~ 19

Author(s):

CAMILLA BETTI ◽

CHIARA LICO ◽

DARIO MAFFI ◽

SIMONE D'ANGELI ◽

MARIA MADDALENA ALTAMURA ◽

...

Keyword(s):

Coat Protein ◽

Potato Virus ◽

Nicotiana Benthamiana ◽

Potato Virus X ◽

Protein Variants

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Molecular characterization of the interaction between the N-terminal region of Potato virus X (PVX) coat protein (CP) and Nicotiana benthamiana PVX CP-interacting protein, NbPCIP1

Virus Genes ◽

10.1007/s11262-013-0896-0 ◽

2013 ◽

Vol 46 (3) ◽

pp. 517-523 ◽

Cited By ~ 5

Author(s):

Mi-Ri Park ◽

Kook-Hyung Kim

Keyword(s):

Coat Protein ◽

Molecular Characterization ◽

Potato Virus ◽

Nicotiana Benthamiana ◽

Potato Virus X ◽

Terminal Region ◽

Interacting Protein

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Nicotiana benthamiana protein, NbPCIP1, interacting with Potato virus X coat protein plays a role as susceptible factor for viral infection

Virology ◽

10.1016/j.virol.2008.12.044 ◽

2009 ◽

Vol 386 (2) ◽

pp. 257-269 ◽

Cited By ~ 29

Author(s):

Mi-Ri Park ◽

Sang-Ho Park ◽

Sang-Yun Cho ◽

Kook-Hyung Kim

Keyword(s):

Coat Protein ◽

Viral Infection ◽

Potato Virus ◽

Nicotiana Benthamiana ◽

Potato Virus X

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Topical Application of Escherichia coli-Encapsulated dsRNA Induces Resistance in Nicotiana benthamiana to Potato Viruses and Involves RDR6 and Combined Activities of DCL2 and DCL4

Plants ◽

10.3390/plants10040644 ◽

2021 ◽

Vol 10 (4) ◽

pp. 644

Author(s):

Khouloud Necira ◽

Mongia Makki ◽

Eugenio Sanz-García ◽

Tomás Canto ◽

Fattouma Djilani-Khouadja ◽

...

Keyword(s):

Escherichia Coli ◽

Virus Infection ◽

Topical Application ◽

Potato Virus ◽

Nicotiana Benthamiana ◽

Fluorescent Protein ◽

Potato Virus X ◽

Dual Infection ◽

Attractive Alternative ◽

Green Fluorescent

Exogenous application of double-stranded RNAs (dsRNAs) for inducing virus resistance in plants represents an attractive alternative to transgene-based silencing approaches. However, improvement of dsRNA stability in natural conditions is required in order to provide long-term protection against the targeted virus. Here, we tested the protective effect of topical application of Escherichia coli-encapsulated dsRNA compared to naked dsRNA against single and dual infection by Potato virus X expressing the green fluorescent protein (PVX-GFP) and Potato virus Y (PVY) in Nicotiana benthamiana. We found that, in our conditions, the effectiveness of E. coli-encapsulated dsRNA in providing RNAi-mediated protection did not differ from that of naked dsRNA. dsRNA vaccination was partly effective against a dual infection by PVX-GFP and PVY, manifested by a delay in the expression of the synergistic symptoms at early times after inoculation. Using PVX-GFP as a reporter virus together with a suite of RNAi knockdown transgenic lines, we have also shown that RNA-directed RNA polymerase 6 and the combined activities of DICER-like 2 (DCL2) and DCL4 act to promote efficient resistance to virus infection conferred by topical application of dsRNA in N. benthamiana. Our results provide evidence that exogenous dsRNA molecules are processed by the RNA silencing pathways commonly used by the host in response to virus infection.

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