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 Transient Coexpression of Individual Genes Encoded by the Triple Gene Block of Potato mop-top virus Reveals Requirements for TGBp1 Trafficking

2004 ◽  
Vol 17 (8) ◽  
pp. 921-930 ◽  
Author(s):  
Andrey A. Zamyatnin ◽  
Andrey G. Solovyev ◽  
Eugene I. Savenkov ◽  
Anna Germundsson ◽  
Maria Sandgren ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Triple Gene Block ◽  
Terminal Region ◽  
Virus Movement ◽  
Cell Periphery ◽  
Movement Proteins ◽  
Gene Block ◽  
Potato Mop Top Virus ◽  
Green Fluorescent

TGBp1, TGBp2, and TGBp3, three plant virus movement proteins encoded by the “triple gene block” (TGB), may act in concert to facilitate cell-to-cell transport of viral RNA genomes. Transient expression of Potato mop-top virus (genus Pomovirus) movement proteins was used as a model to reconstruct interactions between TGB proteins. In bombarded epidermal cells of Nicotiana benthamiana, green fluorescent protein (GFP)-TGBp1 was distributed uniformly. However, in the presence of TGBp2 and TGBp3, GFP-TGBp1 was directed to intermediate bodies at the cell periphery, and to cell wall-embedded punctate bodies. Moreover, GFP-TGBp1 migrated into cells immediately adjacent to the bombarded cell. These data suggest that TGBp2 and TGBp3 mediate transport of GFP-TGBp1 to and through plasmodesmata. Mutagenesis of TGBp1 suggested that the NTPase and helicase activities of TGBp1 were not required for its transport to intermediate bodies directed by TGBp2 and TGBp3, but these activities were essential for the protein association with cell wall-embedded punctate bodies and translocation of TGBp1 to neighboring cells. The C-terminal region of TGBp1 was critical for trafficking mediated by TGBp2 and TGBp3. Mutation analysis also suggested an involvement of the TGBp2 C-terminal region in interactions with TGBp1.

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 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 Microscopic Analysis of Severe Structural Rearrangements of the Plant Endoplasmic Reticulum and Golgi Caused by Overexpression ofPoa semilatent virusMovement Protein

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Andrey G. Solovyev ◽  
Joachim Schiemann ◽  
Sergey Y. Morozov
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Triple Gene Block ◽  
Microscopic Analysis ◽  
Potato Virus X ◽  
Plant Viruses ◽  
Genomic Rnas ◽  
Movement Proteins ◽  
Gene Block ◽  
Ribonucleoprotein Complexes

Cell-to-cell transport of plant viruses is mediated by virus-encoded movement proteins and occurs through plasmodesmata interconnecting neighboring cells in plant tissues. Three movement proteins coded by the “triple gene block” (TGB) and named TGBp1, TGBp2 and TGBp3 have distinct functions in viral transport. TGBp1 binds viral genomic RNAs to form ribonucleoprotein complexes representing the transport form of viral genome, while TGBp2 and TGBp3 are necessary for intracellular delivery of such complexes to plasmodesmata. Recently, it was revealed that overexpression ofPotato virus XTGBp3 triggers the unfolded protein response mitigating the endoplasmic reticulum (ER) stress leading to cell death if this protein reaches high levels in the ER. Here we report microscopic studies of the influence of thePoa semilatent hordeivirusTGBp3 overexpressed inNicotiana benthamianaepidermal cells by particle bombardment on cell endomembranes and demonstrate that the protein C-terminal transmembrane segment contains a determinant responsible for vesiculation and coalescence of the endoplasmic reticulum and Golgi presumably accompanying the ER stress that can be induced upon high-level TGBp3 expression.

scholarly journals The Potato Virus X TGBp2 Protein Plays Dual Functional Roles in Viral Replication and Movement

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
Xiaoyun Wu ◽  
Jiahui Liu ◽  
Mengzhu Chai ◽  
Jinhui Wang ◽  
Dalong Li ◽  
...  
Keyword(s):  
Triple Gene Block ◽  
Potato Virus X ◽  
Plant Viruses ◽  
Open Reading Frames ◽  
Movement Proteins ◽  
Gene Block ◽  
Functional Roles ◽  
Viral Rdrp ◽  
Dual Functional ◽  
Intercellular Movement

ABSTRACTPlant viruses usually encode one or more movement proteins (MP) to accomplish their intercellular movement. A group of positive-strand RNA plant viruses requires three viral proteins (TGBp1, TGBp2, and TGBp3) that are encoded by an evolutionarily conserved genetic module of three partially overlapping open reading frames (ORFs), termed the triple gene block (TGB). However, how these three viral movement proteins function cooperatively in viral intercellular movement is still elusive. Using a novelin vivodouble-stranded RNA (dsRNA) labeling system, we showed that the dsRNAs generated by potato virus X (PVX) RNA-dependent RNA polymerase (RdRp) are colocalized with viral RdRp, which are further tightly covered by “chain mail”-like TGBp2 aggregates and localizes alongside TGBp3 aggregates. We also discovered that TGBp2 interacts with the C-terminal domain of PVX RdRp, and this interaction is required for the localization of TGBp3 and itself to the RdRp/dsRNA bodies. Moreover, we reveal that the central and C-terminal hydrophilic domains of TGBp2 are required to interact with viral RdRp. Finally, we demonstrate that knockout of the entire TGBp2 or the domain involved in interacting with viral RdRp attenuates both PVX replication and movement. Collectively, these findings suggest that TGBp2 plays dual functional roles in PVX replication and intercellular movement.IMPORTANCEMany plant viruses contain three partially overlapping open reading frames (ORFs), termed the triple gene block (TGB), for intercellular movement. However, how the corresponding three proteins coordinate their functions remains obscure. In the present study, we provided multiple lines of evidence supporting the notion that PVX TGBp2 functions as the molecular adaptor bridging the interaction between the RdRp/dsRNA body and TGBp3 by forming “chain mail”-like structures in the RdRp/dsRNA body, which can also enhance viral replication. Taken together, our results provide new insights into the replication and movement of PVX and possibly also other TGB-containing plant viruses.

scholarly journals P42 Movement Protein of Beet necrotic yellow vein virus Is Targeted by the Movement Proteins P13 and P15 to Punctate Bodies Associated with Plasmodesmata

2000 ◽  
Vol 13 (5) ◽  
pp. 520-528 ◽  
Author(s):  
M. Erhardt ◽  
M. Morant ◽  
C. Ritzenthaler ◽  
C. Stussi-Garaud ◽  
H. Guilley ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Triple Gene Block ◽  
Point Mutations ◽  
Cell Movement ◽  
Leading Edge ◽  
Yellow Vein ◽  
Body Formation ◽  
Movement Proteins ◽  
Gene Block

Cell-to-cell movement of Beet necrotic yellow vein virus (BNYVV) is driven by a set of three movement proteins—P42, P13, and P15—organized into a triple gene block (TGB) on viral RNA 2. The first TGB protein, P42, has been fused to the green fluorescent protein (GFP) and fusion proteins between P42 and GFP were expressed from a BNYVV RNA 3-based replicon during virus infection. GFP-P42, in which the GFP was fused to the P42 N terminus, could drive viral cell-to-cell movement when the copy of the P42 gene on RNA 2 was disabled but the C-terminal fusion P42-GFP could not. Confocal microscopy of epidermal cells of Chenopodium quinoa near the leading edge of the infection revealed that GFP-P42 localized to punctate bodies apposed to the cell wall whereas free GFP, expressed from the replicon, was distributed uniformly throughout the cytoplasm. The punctate bodies sometimes appeared to traverse the cell wall or to form pairs of disconnected bodies on each side. The punctate bodies co-localized with callose, indicating that they are associated with plasmodesmata-rich regions such as pit fields. Point mutations in P42 that inhibited its ability to drive cell-to-cell movement also inhibited GFP-P42 punctate body formation. GFP-P42 punctate body formation was dependent on expression of P13 and P15 during the infection, indicating that these proteins act together or sequentially to localize P42 to the plasmodesmata.

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 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 Interactions of the TGB1 Protein during Cell-to-Cell Movement of Barley Stripe Mosaic Virus

2001 ◽  
Vol 75 (18) ◽  
pp. 8712-8723 ◽  
Author(s):  
Diane M. Lawrence ◽  
A. O. Jackson
Keyword(s):  
Subcellular Localization ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Triple Gene Block ◽  
Cell Movement ◽  
Barley Stripe Mosaic Virus ◽  
Helicase Domain ◽  
Chenopodium Amaranticolor ◽  
Gene Block ◽  
Green Fluorescent

ABSTRACT We have recently used a green fluorescent protein (GFP) fusion to the γb protein of Barley stripe mosaic virus (BSMV) to monitor cell-to-cell and systemic virus movement. The γb protein is involved in expression of the triple gene block (TGB) proteins encoded by RNAβ but is not essential for cell-to-cell movement. The GFP fusion appears not to compromise replication or movement substantially, and mutagenesis experiments demonstrated that the three most abundant TGB-encoded proteins, βb (TGB1), βc (TGB3), and βd (TGB2), are each required for cell-to-cell movement (D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65–75, 2001). We have now extended these analyses by engineering a fusion of GFP to TGB1 to examine the expression and interactions of this protein during infection. BSMV derivatives containing the TGB1 fusion were able to move from cell to cell and establish local lesions in Chenopodium amaranticolor and systemic infections of Nicotiana benthamiana and barley. In these hosts, the GFP-TGB1 fusion protein exhibited a temporal pattern of expression along the advancing edge of the infection front. Microscopic examination of the subcellular localization of the GFP-TGB1 protein indicated an association with the endoplasmic reticulum and with plasmodesmata. The subcellular localization of the TGB1 protein was altered in infections in which site-specific mutations were introduced into the six conserved regions of the helicase domain and in mutants unable to express the TGB2 and/or TGB3 proteins. These results are compatible with a model suggesting that movement requires associations of the TGB1 protein with cytoplasmic membranes that are facilitated by the TGB2 and TGB3 proteins.

scholarly journals Replication and trafficking of a plant virus are coupled at the entrances of plasmodesmata

2013 ◽  
Vol 201 (7) ◽  
pp. 981-995 ◽  
Author(s):  
Jens Tilsner ◽  
Olga Linnik ◽  
Marion Louveaux ◽  
Ian M. Roberts ◽  
Sean N. Chapman ◽  
...  
Keyword(s):  
Plant Virus ◽  
Plant Cell Wall ◽  
Triple Gene Block ◽  
Potato Virus X ◽  
Plant Viruses ◽  
Viral Coat Protein ◽  
Movement Proteins ◽  
Gene Block ◽  
Replication Sites ◽  
Viral Coat

Plant viruses use movement proteins (MPs) to modify intercellular pores called plasmodesmata (PD) to cross the plant cell wall. Many viruses encode a conserved set of three MPs, known as the triple gene block (TGB), typified by Potato virus X (PVX). In this paper, using live-cell imaging of viral RNA (vRNA) and virus-encoded proteins, we show that the TGB proteins have distinct functions during movement. TGB2 and TGB3 established endoplasmic reticulum–derived membranous caps at PD orifices. These caps harbored the PVX replicase and nonencapsidated vRNA and represented PD-anchored viral replication sites. TGB1 mediated insertion of the viral coat protein into PD, probably by its interaction with the 5′ end of nascent virions, and was recruited to PD by the TGB2/3 complex. We propose a new model of plant virus movement, which we term coreplicational insertion, in which MPs function to compartmentalize replication complexes at PD for localized RNA synthesis and directional trafficking of the virus between cells.

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