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 Cell-to-Cell Movement of Potato virus X: The Role of p12 and p8 Encoded by the Second and Third Open Reading Frames of the Triple Gene Block

2001 ◽  
Vol 14 (10) ◽  
pp. 1158-1167 ◽  
Author(s):  
Atsushi Tamai ◽  
Tetsuo Meshi
Keyword(s):  
Potato Virus ◽  
Fluorescent Protein ◽  
Microprojectile Bombardment ◽  
Triple Gene Block ◽  
Cell Movement ◽  
Potato Virus X ◽  
Open Reading Frames ◽  
Gene Block ◽  
Infected Cells ◽  
Green Fluorescent

Potato virus X (PVX) requires three proteins, p25, p12, and p8, encoded by the triple gene block plus the coat protein (CP) for cell-to-cell movement. When each of these proteins was co-expressed with a cytosolic green fluorescent protein (GFP) in the epidermal cells of Nicotiana benthamiana by the microprojectile bombardment-mediated gene delivery method, only p12 enhanced diffusion of co-expressed GFP, indicating an ability to alter plasmodesmal permeability. p25, p12, and CP, expressed transiently in the initially infected cells, transcomplemented the corresponding movement-defective mutants to spread through two or more cell boundaries. Thus, these proteins probably move from cell to cell with the genomic RNA. In contrast, p8 only functioned intracellularly and was not absolutely required for cell-to-cell movement. Since overexpression of p12 overcame the p8 deficiency, p8 appears to facilitate the functioning of p12, presumably by mediating its intracellular trafficking. Considering the likelihood that p12 and p8 are membrane proteins, it is suggested that intercellular as well as intracellular movement of PVX involves a membrane-mediated process.

scholarly journals Cell-to-Cell Movement of the 25K Protein of Potato virus X Is Regulated by Three Other Viral Proteins

2000 ◽  
Vol 13 (6) ◽  
pp. 599-605 ◽  
Author(s):  
Yang Yang ◽  
Biao Ding ◽  
David C. Baulcombe ◽  
Jeanmarie Verchot
Keyword(s):  
Transgenic Tobacco ◽  
Potato Virus ◽  
Fluorescent Protein ◽  
Cell Movement ◽  
Potato Virus X ◽  
Viral Proteins ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Green Fluorescent ◽  
Potential Interactions

The 25K, 12K, and 8K proteins and coat protein (CP) of Potato virus X (PVX) are required for virus cell-to-cell movement. In this study, experiments were conducted to determine whether the PVX 25K protein moves cell to cell and to explore potential interactions between the PVX 25K, 12K, and 8K proteins and CP. The PVX 25K gene was fused to the green fluorescent protein (GFP) gene and inserted into plasmids adjacent to the cauliflower mosaic virus 35S promoter. These plasmids were introduced by biolistic bombardment to transgenic tobacco expressing the PVX 12K, 8K, and CP genes. The GFP:25K fused proteins moved cell to cell on nontransgenic tobacco and tobacco expressing either the 12K or 8K proteins. However, the GFP:25K proteins did not move on transgenic tobacco expressing the combined 12K/8K genes or the CP gene. Thus, movement of the PVX 25K protein through plas-modesmata may be regulated by interactions with other PVX proteins.

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 Fluorescently Tagged Potato virus Y: A Versatile Tool for Functional Analysis of Plant-Virus Interactions

2015 ◽  
Vol 28 (7) ◽  
pp. 739-750 ◽  
Author(s):  
Matevz Rupar ◽  
Florence Faurez ◽  
Michel Tribodet ◽  
Ion Gutiérrez-Aguirre ◽  
Agnès Delaunay ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Potato Virus ◽  
Plant Virus ◽  
Potato Virus Y ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Movement ◽  
Long Distance ◽  
Green Fluorescent

Potato virus Y (PVY) is an economically important plant virus that infects Solanaceous crops such as tobacco and potato. To date, studies into the localization and movement of PVY in plants have been limited to detection of viral RNA or proteins ex vivo. Here, a PVY N605 isolate was tagged with green fluorescent protein (GFP), characterized and used for in vivo tracking. In Nicotiana tabacum cv. Xanthi, PVY N605-GFP was biologically comparable to nontagged PVY N605, stable through three plant-to-plant passages and persisted for four months in infected plants. GFP was detected before symptoms and fluorescence intensity correlated with PVY RNA concentrations. PVY N605-GFP provided in vivo tracking of long-distance movement, allowing estimation of the cell-to-cell movement rate of PVY in N. tabacum cv. Xanthi (7.1 ± 1.5 cells per hour). PVY N605-GFP was adequately stable in Solanum tuberosum cvs. Désirée and NahG-Désirée and able to infect S. tuberosum cvs. Bintje and Bea, Nicotiana benthamiana, and wild potato relatives. PVY N605-GFP is therefore a powerful tool for future studies of PVY-host interactions, such as functional analysis of viral and plant genes involved in viral movement.

scholarly journals Intracellular Targeting of a Hordeiviral Membrane-Spanning Movement Protein: Sequence Requirements and Involvement of an Unconventional Mechanism

2007 ◽  
Vol 82 (3) ◽  
pp. 1284-1293 ◽  
Author(s):  
Mikhail V. Schepetilnikov ◽  
Andrey G. Solovyev ◽  
Elena N. Gorshkova ◽  
Joachim Schiemann ◽  
Alexey I. Prokhnevsky ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Detectable Effect ◽  
Movement Proteins ◽  
Membrane Spanning ◽  
Green Fluorescent

ABSTRACT The membrane-spanning protein TGBp3 is one of the three movement proteins (MPs) of Poa semilatent virus. TGBp3 is thought to direct other viral MPs and genomic RNA to peripheral bodies located in close proximity to plasmodesmata. We used the ectopic expression of green fluorescent protein-fused TGBp3 in epidermal cells of Nicotiana benthamiana leaves to study the TGBp3 intracellular trafficking pathway. Treatment with inhibitors was used to reveal that the targeting of TGBp3 to plasmodesmata does not require a functional cytoskeleton or secretory system. In addition, the suppression of endoplasmic reticulum-derived vesicle formation by a dominant negative mutant of small GTPase Sar1 had no detectable effect on TGBp3 trafficking to peripheral bodies. Collectively, these results suggested the involvement of an unconventional pathway in the intracellular transport of TGBp3. The determinants of targeting to plasmodesmata were localized to the C-terminal region of TGBp3, including the conserved hydrophilic and terminal membrane-spanning domains.

scholarly journals A Versatile Assay for the Identification of RNA Silencing Suppressors Based on Complementation of Viral Movement

2008 ◽  
Vol 21 (7) ◽  
pp. 879-890 ◽  
Author(s):  
Jason G. Powers ◽  
Tim L. Sit ◽  
Feng Qu ◽  
T. Jack Morris ◽  
Kook-Hyung Kim ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Rna Silencing ◽  
Fluorescent Protein ◽  
Wide Spectrum ◽  
Cell Movement ◽  
Suppressor Activity ◽  
Reading Frame ◽  
Movement Proteins ◽  
In Trans ◽  
Green Fluorescent

The cell-to-cell movement of Turnip crinkle virus (TCV) in Nicotiana benthamiana requires the presence of its coat protein (CP), a known suppressor of RNA silencing. RNA transcripts of a TCV construct containing a reporter gene (green fluorescent protein) (TCV-sGFP) in place of the CP open reading frame generated foci of three to five cells. TCV CP delivered in trans by Agrobacterium tumefaciens infiltration potentiated movement of TCV-sGFP and increased foci diameter, on average, by a factor of four. Deletion of the TCV movement proteins in TCV-sGFP (construct TCVΔ92-sGFP) abolished the movement complementation ability of TCV CP. Other known suppressors of RNA silencing from a wide spectrum of viruses also complemented the movement of TCV-sGFP when delivered in trans by Agrobacterium tumefaciens. These include suppressors from nonplant viruses with no known plant movement function, demonstrating that this assay is based solely on RNA silencing suppression. While the TCV-sGFP construct is primarily used as an infectious RNA transcript, it was also subcloned for direct expression from Agrobacterium tumefaciens for simple quantification of suppressor activity based on fluorescence levels in whole leaves. Thus, this system provides the flexibility to assay for suppressor activity in either the cytoplasm or nucleus, depending on the construct employed.

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 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.

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