Production of Antimicrobial Defensin in Nicotiana benthamiana with a Potato Virus X Vector

A recombinant plasmid, pTXS.TH, was constructed to express the gene-encoding wasabi (Wasabia japonica) defensin with the potato virus X (PVX) vector. pTXS.TH allows the expression of defensin in the host Nicotiana benthamiana, and the defensin protein WT1 can be purified from virus-infected leaves by heat treatment and affinity chromatography. WT1 exhibits strong antifungal activity toward the phytopathogenic fungi Magnaporthe grisea(50% inhibitory concentration [IC50] = 5 μg/ml) and Botrytis cinerea (IC50 = 20 μg/ml) but is weakly active against the phytopathogenic bacterium Pseudomonas cichorii. This virus-mediated expression system is a rapid and efficient method to produce and characterize antimicrobial proteins in plants. It is particularly useful for the study of proteins that are difficult to produce with other expression systems.

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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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Interplay between potato virus X and RNA granules in Nicotiana benthamiana

Virus Research ◽

10.1016/j.virusres.2019.197823 ◽

2020 ◽

Vol 276 ◽

pp. 197823

Author(s):

Gabriel Robles-Luna ◽

Nicolás Furman ◽

María Florencia Barbarich ◽

Nicolás Carlotto ◽

Alejandra Attorresi ◽

...

Keyword(s):

Potato Virus ◽

Nicotiana Benthamiana ◽

Potato Virus X ◽

Rna Granules

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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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Soluble, Template-Dependent Extracts from Nicotiana benthamiana Plants Infected with Potato Virus X Transcribe both Plus- and Minus-Strand RNA Templates

Virology ◽

10.1006/viro.2000.0512 ◽

2000 ◽

Vol 275 (2) ◽

pp. 444-451 ◽

Cited By ~ 17

Author(s):

Carol A. Plante ◽

Kook-Hyung Kim ◽

Neeta Pillai-Nair ◽

Toba A.M. Osman ◽

Kenneth W. Buck ◽

...

Keyword(s):

Potato Virus ◽

Nicotiana Benthamiana ◽

Potato Virus X ◽

Minus Strand

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Production of an Engineered Killer Peptide in Nicotiana benthamiana by Using a Potato virus X Expression System

Applied and Environmental Microbiology ◽

10.1128/aem.71.10.6360-6367.2005 ◽

2005 ◽

Vol 71 (10) ◽

pp. 6360-6367 ◽

Cited By ~ 27

Author(s):

Marcello Donini ◽

Chiara Lico ◽

Selene Baschieri ◽

Stefania Conti ◽

Walter Magliani ◽

...

Keyword(s):

Antimicrobial Activity ◽

Pseudomonas Syringae ◽

Potato Virus ◽

Broad Spectrum ◽

Plant Pathogens ◽

Expression System ◽

Potato Virus X ◽

Scale Production ◽

Human Pathogens ◽

Single Chain

ABSTRACT The decapeptide killer peptide (KP) derived from the sequence of a single-chain, anti-idiotypic antibody acting as a functional internal image of a microbicidal, broad-spectrum yeast killer toxin (KT) was shown to exert a strong microbicidal activity against human pathogens. With the aim to exploit this peptide to confer resistance to plant pathogens, we assayed its antimicrobial activity against a broad spectrum of phytopathogenic bacteria and fungi. Synthetic KP exhibited antimicrobial activity in vitro towards Pseudomonas syringae, Erwinia carotovora, Botrytis cinerea, and Fusarium oxysporum. KP was also expressed in plants by using a Potato virus X (PVX)-derived vector as a fusion to the viral coat protein, yielding chimeric virus particles (CVPs) displaying the heterologous peptide. Purified CVPs showed enhanced antimicrobial activity against the above-mentioned plant pathogens and human pathogens such as Staphylococcus aureus and Candida albicans. Moreover, in vivo assays designed to challenge KP-expressing plants (as CVPs) with Pseudomonas syringae pv. tabaci showed enhanced resistance to bacterial attack. The results indicate that the PVX-based display system is a high-yield, rapid, and efficient method to produce and evaluate antimicrobial peptides in plants, representing a milestone for the large-scale production of high-added-value peptides through molecular farming. Moreover, KP is a promising molecule to be stably engineered in plants to confer broad-spectrum resistance to phytopathogens.

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