scholarly journals Identification of a Movement Protein of the Tenuivirus Rice Stripe Virus

2008 ◽  
Vol 82 (24) ◽  
pp. 12304-12311 ◽  
Author(s):  
Ruyi Xiong ◽  
Jianxiang Wu ◽  
Yijun Zhou ◽  
Xueping Zhou
Keyword(s):  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Severe Disease ◽  
Rice Stripe Virus ◽  
Potato Virus X ◽  
Infected Leaf ◽  
Biolistic Bombardment ◽  
Movement Proteins ◽  
Green Fluorescent

ABSTRACT Rice stripe virus (RSV) is the type member of the genus Tenuivirus. RSV has four single-stranded RNAs and causes severe disease in rice fields in different parts of China. To date, no reports have described how RSV spreads within host plants or the viral and/or host factor(s) required for tenuivirus movement. We investigated functions of six RSV-encoded proteins using trans-complementation experiments and biolistic bombardment. We demonstrate that NSvc4, encoded by RSV RNA4, supports the intercellular trafficking of a movement-deficient Potato virus X in Nicotiana benthamiana leaves. We also determined that upon biolistic bombardment or agroinfiltration, NSvc4:enhanced green fluorescent protein (eGFP) fusion proteins localize predominantly near or within the walls of onion and tobacco epidermal cells. In addition, the NSvc4:eGFP fusion protein can move from initially bombarded cells to neighboring cells in Nicotiana benthamiana leaves. Immunocytochemistry using tissue sections from RSV-infected rice leaves and an RSV NSvc4-specific antibody showed that the NSvc4 protein accumulated in walls of RSV-infected leaf cells. Gel retardation assays revealed that the NSvc4 protein interacts with single-stranded RNA in vitro, a common feature of many reported plant viral movement proteins (MPs). RSV NSvc4 failed to interact with the RSV nucleocapsid protein using yeast two-hybrid assays. Taken together, our data indicate that RSV NSvc4 is likely an MP of the virus. This is the first report describing a tenuivirus MP.

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

2008 ◽  
Vol 21 (2) ◽  
pp. 178-187 ◽  
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.

scholarly journals The hydrophobic segment of Potato virus X TGBp3 is a major determinant of the protein intracellular trafficking

2005 ◽  
Vol 86 (8) ◽  
pp. 2379-2391 ◽  
Author(s):  
M. V. Schepetilnikov ◽  
U. Manske ◽  
A. G. Solovyev ◽  
A. A. Zamyatnin ◽  
J. Schiemann ◽  
...  
Keyword(s):  
Potato Virus ◽  
Intracellular Trafficking ◽  
Intracellular Transport ◽  
Fluorescent Protein ◽  
Cell Movement ◽  
Potato Virus X ◽  
Movement Proteins ◽  
C Terminus ◽  
Hydrophilic Region ◽  
Green Fluorescent

Potato virus X (PVX) encodes three movement proteins, TGBp1, TGBp2 and TGBp3. The 8 kDa TGBp3 is a membrane-embedded protein that has an N-terminal hydrophobic sequence segment and a hydrophilic C terminus. TGBp3 mutants with deletions in the C-terminal hydrophilic region retain the ability to be targeted to cell peripheral structures and to support limited PVX cell-to-cell movement, suggesting that the basic TGBp3 functions are associated with its N-terminal transmembrane region. Fusion of green fluorescent protein to the TGBp3 N terminus abrogates protein activities in intracellular trafficking and virus movement. The intracellular transport of TGBp3 from sites of its synthesis in the rough endoplasmic reticulum (ER) to ER-derived peripheral bodies involves a non-conventional COPII-independent pathway. However, integrity of the C-terminal hydrophilic sequence is required for entrance to this non-canonical route.

scholarly journals ORF6 of Tobacco mosaic virus is a determinant of viral pathogenicity in Nicotiana benthamiana

2004 ◽  
Vol 85 (10) ◽  
pp. 3123-3133 ◽  
Author(s):  
Tomas Canto ◽  
Stuart A. MacFarlane ◽  
Peter Palukaitis
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Tobacco Rattle Virus ◽  
Potato Virus X ◽  
Host Responses ◽  
Reading Frame ◽  
Virus Accumulation ◽  
Green Fluorescent

Tobacco mosaic virus (TMV) contains a sixth open reading frame (ORF6) that potentially encodes a 4·8 kDa protein. Elimination of ORF6 from TMV attenuated host responses in Nicotiana benthamiana without alteration in virus accumulation. Furthermore, heterologous expression of TMV ORF6 from either potato virus X (PVX) or tobacco rattle virus (TRV) vectors enhanced the virulence of both viruses in N. benthamiana, also without effects on their accumulation. By contrast, the presence or absence of TMV ORF6 had no effect on host response or virus accumulation in N. tabacum plants infected with TMV or PVX. TMV ORF6 also had no effect on the synergism between TMV and PVX in N. tabacum. However, the presence of the TMV ORF6 did have an effect on the pathogenicity of a TRV vector in N. tabacum. In three different types of assay carried out in N. benthamiana plants, expression of TMV ORF6 failed to suppress gene silencing. Expression in N. benthamiana epidermal cells of the encoded 4·8 kDa protein fused to the green fluorescent protein at either end showed, in addition to widespread cytosolic fluorescence, plasmodesmatal targeting specific to both fusion constructs. The role of the ORF6 in host responses is discussed.

scholarly journals Acibenzolar-S-Methyl Restricts Infection of Nicotiana benthamiana by Plantago Asiatica Mosaic Virus at Two Distinct Stages

2019 ◽  
Vol 32 (11) ◽  
pp. 1475-1486 ◽  
Author(s):  
Yuki Matsuo ◽  
Fawzia Novianti ◽  
Miki Takehara ◽  
Toshiyuki Fukuhara ◽  
Tsutomu Arie ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Potato Virus X ◽  
Defense Responses ◽  
Plant Viruses ◽  
Long Distance ◽  
Plantago Asiatica ◽  
Green Fluorescent ◽  
Long Distance Movement

Plant activators, including acibenzolar-S-methyl (ASM), are chemical compounds that stimulate plant defense responses to pathogens. ASM treatment inhibits infection by a variety of plant viruses, however, the mechanisms of this broad-spectrum and strong effect remain poorly understood. We employed green fluorescent protein (GFP)-expressing viruses and Nicotiana benthamiana plants to identify the infection stages that are restricted by ASM. ASM suppressed infection by three viral species, plantago asiatica mosaic virus (PlAMV), potato virus X (PVX), and turnip mosaic virus (TuMV), in inoculated cells. Furthermore, ASM delayed the long-distance movement of PlAMV and PVX, and the cell-to-cell (short range) movement of TuMV. The ASM-mediated delay of long-distance movement of PlAMV was not due to the suppression of viral accumulation in the inoculated leaves, indicating that ASM restricts PlAMV infection in at least two independent steps. We used Arabidopsis thaliana mutants to show that the ASM-mediated restriction of PlAMV infection requires the NPR1 gene but was independent of the dicer-like genes essential for RNA silencing. Furthermore, experiments using protoplasts showed that ASM treatment inhibited PlAMV replication without cell death. Our approach, using GFP-expressing viruses, will be useful for the analysis of mechanisms underlying plant activator–mediated virus restriction.

scholarly journals Expression, localization and effects on virulence of the cysteine-rich 8 kDa protein of Potato mop-top virus

2005 ◽  
Vol 86 (10) ◽  
pp. 2879-2889 ◽  
Author(s):  
N. I. Lukhovitskaya ◽  
N. E. Yelina ◽  
A. A. Zamyatnin ◽  
M. V. Schepetilnikov ◽  
A. G. Solovyev ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescent Protein ◽  
Triple Gene Block ◽  
Potato Virus X ◽  
Host Responses ◽  
Reading Frame ◽  
Movement Proteins ◽  
Gene Block ◽  
Potato Mop Top Virus ◽  
Green Fluorescent

Potato mop-top virus (PMTV) RNA3 contains a triple gene block (TGB) encoding viral movement proteins and an open reading frame for a putative 8 kDa cysteine-rich protein (CRP). In this study, PMTV CRP was shown to be expressed in the course of virus infection, and a PMTV CRP-specific subgenomic RNA was mapped. CRP has previously been shown to be dispensable for infection of PMTV in Nicotiana benthamiana. In this study, PMTV CRP was found to increase the severity of disease symptoms when expressed from Potato virus X or Tobacco mosaic virus in N. benthamiana and Nicotiana tabacum, suggesting that the protein affects virulence of the virus or might suppress a host defence mechanism. However, PMTV CRP did not show RNA silencing suppression activity in three assays. Host responses to the PMTV CRP expression from different viral genomes ranged from an absence of response to extreme resistance at a single cell level and were dependent on the viral genome. These findings emphasized involvement of viral proteins and/or virus-induced cell components in the plant reaction to CRP. PMTV CRP was predicted to possess a transmembrane segment. CRP fused to the green fluorescent protein was associated with endoplasmic reticulum-derived membranes and induced dramatic rearrangements of the endoplasmic reticulum structure, which might account for protein functions as a virulence factor of the virus.

scholarly journals Rice yellow stunt rhabdovirus Protein 6 Suppresses Systemic RNA Silencing by Blocking RDR6-Mediated Secondary siRNA Synthesis

2013 ◽  
Vol 26 (8) ◽  
pp. 927-936 ◽  
Author(s):  
Hongyan Guo ◽  
Xiaoguang Song ◽  
Chuanmiao Xie ◽  
Yan Huo ◽  
Fujie Zhang ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Fluorescent Protein ◽  
Rice Stripe Virus ◽  
Potato Virus X ◽  
Structural Protein ◽  
Susceptibility To Infection ◽  
Exact Function ◽  
Secondary Sirna

The P6 protein of Rice yellow stunt rhabdovirus (RYSV) is a virion structural protein that can be phosphorylated in vitro. However its exact function remains elusive. We found that P6 enhanced the virulence of Potato virus X (PVX) in Nicotiana benthamiana and N. tabacum plants, suggesting that it might function as a suppressor of RNA silencing. We examined the mechanism of P6-mediated silencing suppression by transiently expressing P6 in both N. benthamiana leaves and rice protoplasts. Our results showed that P6 could repress the production of secondary siRNAs and inhibit systemic green fluorescent protein RNA silencing but did not interfere with local RNA silencing in N. benthamiana plants or in rice protoplasts. Intriguingly, P6 and RDR6 had overlapping subcellular localization and P6 bound both rice and Arabidopsis RDR6 in vivo. Furthermore, transgenic rice plants expressing P6 showed enhanced susceptibility to infection by Rice stripe virus. Hence, we propose that P6 is part of the RYSV's counter-defense machinery against the plant RNA silencing system and plays a role mainly in affecting RDR6-mediated secondary siRNA synthesis. Our work provides a new perspective on how a plant-infecting nucleorhabdovirus may counteract host RNA silencing-mediated antiviral defense.

scholarly journals 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 ◽  
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.

scholarly journals Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

2021 ◽  
Vol 9 (5) ◽  
pp. 1005
Author(s):  
Olga Chervyakova ◽  
Elmira Tailakova ◽  
Nurlan Kozhabergenov ◽  
Sandugash Sadikaliyeva ◽  
Kulyaisan Sultankulova ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Viral Vector ◽  
Foot And Mouth Disease ◽  
Open Reading Frames ◽  
Foreign Gene ◽  
Mouth Disease Virus ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
Reading Frames

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 379
Author(s):  
Breanne M. Head ◽  
Christopher I. Graham ◽  
Teassa MacMartin ◽  
Yoav Keynan ◽  
Ann Karen C. Brassinga
Keyword(s):  
Growth Kinetics ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Disease Incidence ◽  
Acanthamoeba Castellanii ◽  
Infection Model ◽  
Intracellular Infection ◽  
Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

scholarly journals CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Peng-Fei Fu ◽  
Xuan Cheng ◽  
Bing-Qian Su ◽  
Li-Fang Duan ◽  
Cong-Rong Wang ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Antiviral Agents ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Economic Losses ◽  
Green Fluorescent ◽  
Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

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