Movement of potexviruses requires species-specific interactions among the cognate triple gene block proteins, as revealed by a trans-complementation assay based on the bamboo mosaic virus satellite RNA-mediated expression system

The intra- and intercellular transport of potexviruses require interactions among viral RNA, coat protein and elements of the triple gene block proteins (TGBps). In this study, the requirement of bamboo mosaic virus (BaMV) TGBps for movement functions and the compatibilities with those of two potexviruses, Potato virus X (PVX) and Foxtail mosaic virus (FoMV), were examined using a satellite RNA-mediated trans-complementation assay system. Single or multiple TGBps of BaMV, PVX and FoMV were expressed from BaMV satellite RNA (satBaMV RNA) vectors to complement the functions of green fluorescent protein-tagged, movement-defective BaMV with mutation(s) in the matching gene(s). It was found that individual BaMV TGBps expressed from the satellite vector could function normally in trans, whereas bi-gene BaMV TGBp constructs in which the expression of TGBp3 might be impaired and individual TGBp genes from PVX or FoMV could not complement the movement functions of the defective helper viruses. Furthermore, alterations of the ratio among TGBps by ectopic expression of individual components of TGBps from satBaMV RNA vectors did not affect the cell-to-cell movement capabilities of wild-type BaMV significantly. The results indicate that species-specific interactions among movement proteins are obligatory for the cell-to-cell movement of BaMV and possibly other potexviruses.

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Cell-to-Cell Movement of Beet Necrotic Yellow Vein Virus: I. Heterologous Complementation Experiments Provide Evidence for Specific Interactions Among the Triple Gene Block Proteins

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1998.11.7.618 ◽

1998 ◽

Vol 11 (7) ◽

pp. 618-625 ◽

Cited By ~ 44

Author(s):

Emmanuelle Lauber ◽

Claudine Bleykasten-Grosshans ◽

M. Erhardt ◽

S. Bouzoubaa ◽

G. Jonard ◽

...

Keyword(s):

Mosaic Virus ◽

Triple Gene Block ◽

Chenopodium Quinoa ◽

Alfalfa Mosaic Virus ◽

Cell Movement ◽

Yellow Vein ◽

Specific Interactions ◽

In Planta ◽

Movement Proteins ◽

Gene Block

Cell-to-cell movement of beet necrotic yellow vein virus (BNYVV) requires three proteins encoded by a triple gene block (TGB) on viral RNA 2. A BNYVV RNA 3-derived replicon was used to express movement proteins of other viruses and the ability of these proteins to functionally substitute for the BNYVV TGB proteins was tested by coinoculation of TGB-defective BNYVV with the various replicons to Chenopodium quinoa. Trans-heterocomplementation was successful with the movement protein (P30) of tobacco mosaic virus but not with the tubule-forming movement proteins of alfalfa mosaic virus and grapevine fanleaf virus. Trans-complementation of BNYVV movement was also observed when all three TGB proteins of the distantly related peanut clump virus were supplied together but not when they were substituted for their BNYVV counterparts one by one. When P30 was used to drive BNYVV movement in trans, accumulation of the first TGB protein of BNYVV was adversely affected by null mutations in the second and third TGB proteins. Taken together, these results suggest that highly specific interactions among cognate TGB proteins are important for their function and/or stability in planta.

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Triple Gene Block Protein Interactions Involved in Movement of Barley Stripe Mosaic Virus

Journal of Virology ◽

10.1128/jvi.02586-07 ◽

2008 ◽

Vol 82 (10) ◽

pp. 4991-5006 ◽

Cited By ~ 42

Author(s):

Hyoun-Sub Lim ◽

Jennifer N. Bragg ◽

Uma Ganesan ◽

Diane M. Lawrence ◽

Jialin Yu ◽

...

Keyword(s):

Mosaic Virus ◽

Triple Gene Block ◽

Cell Movement ◽

Wild Type Virus ◽

Barley Stripe Mosaic Virus ◽

Wild Type ◽

In Planta ◽

Gene Block ◽

Physical Interactions

ABSTRACT Barley stripe mosaic virus (BSMV) encodes three movement proteins in an overlapping triple gene block (TGB), but little is known about the physical interactions of these proteins. We have characterized a ribonucleoprotein (RNP) complex consisting of the TGB1 protein and plus-sense BSMV RNAs from infected barley plants and have identified TGB1 complexes in planta and in vitro. Homologous TGB1 binding was disrupted by site-specific mutations in each of the first two N-terminal helicase motifs but not by mutations in two C-terminal helicase motifs. The TGB2 and TGB3 proteins were not detected in the RNP, but affinity chromatography and yeast two-hybrid experiments demonstrated that TGB1 binds to TGB3 and that TGB2 and TGB3 form heterologous interactions. These interactions required the TGB2 glycine 40 and the TGB3 isoleucine 108 residues, and BSMV mutants containing these amino acid substitution were unable to move from cell to cell. Infectivity experiments indicated that TGB1 separated on a different genomic RNA from TGB2 and TGB3 could function in limited cell-to-cell movement but that the rates of movement depended on the levels of expression of the proteins and the contexts in which they are expressed. Moreover, elevated expression of the wild-type TGB3 protein interfered with cell-to-cell movement but movement was not affected by the similar expression of a TGB3 mutant that fails to interact with TGB2. These experiments suggest that BSMV movement requires physical interactions of TGB2 and TGB3 and that substantial deviation from the TGB protein ratios expressed by the wild-type virus compromises movement.

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

Journal of Virology ◽

10.1128/jvi.75.18.8712-8723.2001 ◽

2001 ◽

Vol 75 (18) ◽

pp. 8712-8723 ◽

Cited By ~ 49

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.

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Mutation of a chloroplast-targeting signal in Alternanthera mosaic virus TGB3 impairs cell-to-cell movement and eliminates long-distance virus movement

Journal of General Virology ◽

10.1099/vir.0.019448-0 ◽

2010 ◽

Vol 91 (8) ◽

pp. 2102-2115 ◽

Cited By ~ 24

Author(s):

Hyoun-Sub Lim ◽

Anna Maria Vaira ◽

Hanhong Bae ◽

Jennifer N. Bragg ◽

Steven E. Ruzin ◽

...

Keyword(s):

Mosaic Virus ◽

Critical Role ◽

Triple Gene Block ◽

Cell Movement ◽

Potato Virus X ◽

Start Codon ◽

Long Distance ◽

Mesophyll Cells ◽

Chloroplast Targeting ◽

Gene Block

Cell-to-cell movement of potexviruses requires coordinated action of the coat protein and triple gene block (TGB) proteins. The structural properties of Alternanthera mosaic virus (AltMV) TGB3 were examined by methods differentiating between signal peptides and transmembrane domains, and its subcellular localization was studied by Agrobacterium-mediated transient expression and confocal microscopy. Unlike potato virus X (PVX) TGB3, AltMV TGB3 was not associated with the endoplasmic reticulum, and accumulated preferentially in mesophyll cells. Deletion and site-specific mutagenesis revealed an internal signal VL(17,18) of TGB3 essential for chloroplast localization, and either deletion of the TGB3 start codon or alteration of the chloroplast-localization signal limited cell-to-cell movement to the epidermis, yielding a virus that was unable to move into the mesophyll layer. Overexpression of AltMV TGB3 from either AltMV or PVX infectious clones resulted in veinal necrosis and vesiculation at the chloroplast membrane, a cytopathology not observed in wild-type infections. The distinctive mesophyll and chloroplast localization of AltMV TGB3 highlights the critical role played by mesophyll targeting in virus long-distance movement within plants.

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The Two Conserved Cysteine Residues of the Triple Gene Block Protein 2 Are Critical for Both Cell-to-Cell and Systemic Movement of Bamboo mosaic virus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-11-1379 ◽

2009 ◽

Vol 22 (11) ◽

pp. 1379-1388 ◽

Cited By ~ 17

Author(s):

Yang-Hao Tseng ◽

Hsiu-Ting Hsu ◽

Yuan-Lin Chou ◽

Chung-Chi Hu ◽

Na-Sheng Lin ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mosaic Virus ◽

Outer Surface ◽

Transmembrane Protein ◽

Triple Gene Block ◽

Cell Movement ◽

Gene Block ◽

Systemic Movement ◽

Granular Vesicles ◽

Triple Gene Block Protein

The triple gene block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) is a transmembrane protein which is known to be required for the cell-to-cell movement of potexviruses. This protein has two conserved Cys residues, Cys-109 and Cys-112, at its C-terminal tail, which is supposed to be exposed on the outer surface of the endoplasmic reticulum (ER) membrane and ER-derived granular vesicles. In this study, we investigated the importance of these two Cys residues on the cell-to-cell and systemic movement of BaMV. Our results indicate that the Cys-to-Ala substitutions in TGBp2 make the cell-to-cell movement of BaMV relatively inefficient and the systemic movement of BaMV severely inhibited. Moreover, the defect in systemic movement is attributed to the inefficient transport of viral RNA in the phloem of petiole. Clearly, TGBp2 is critical not only for the cell-to-cell but also for the systemic movement of BaMV. In addition, the conserved Cys residues are important for the functioning of TGBp2.

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Subcellular Localization of the Barley Stripe Mosaic Virus Triple Gene Block Proteins

Journal of Virology ◽

10.1128/jvi.01862-09 ◽

2009 ◽

Vol 83 (21) ◽

pp. 11413-11413 ◽

Cited By ~ 1

Author(s):

Hyoun-Sub Lim ◽

Jennifer N. Bragg ◽

Uma Ganesan ◽

Steven Ruzin ◽

Denise Schichnes ◽

...

Keyword(s):

Subcellular Localization ◽

Mosaic Virus ◽

Triple Gene Block ◽

Barley Stripe Mosaic Virus ◽

Gene Block

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A Thioredoxin Domain-Containing Protein Interacts with Pepino mosaic virus Triple Gene Block Protein 1

International Journal of Molecular Sciences ◽

10.3390/ijms19123747 ◽

2018 ◽

Vol 19 (12) ◽

pp. 3747

Author(s):

Matthaios Mathioudakis ◽

Souheyla Khechmar ◽

Carolyn Owen ◽

Vicente Medina ◽

Karima Ben Mansour ◽

...

Keyword(s):

Mosaic Virus ◽

Triple Gene Block ◽

Polyclonal Antiserum ◽

Post Inoculation ◽

Mrna Levels ◽

In Planta ◽

Pepino Mosaic Virus ◽

Gene Block ◽

Triple Gene Block Protein

Pepino mosaic virus (PepMV) is a mechanically-transmitted tomato pathogen of importance worldwide. Interactions between the PepMV coat protein and triple gene block protein (TGBp1) with the host heat shock cognate protein 70 and catalase 1 (CAT1), respectively, have been previously reported by our lab. In this study, a novel tomato interactor (SlTXND9) was shown to bind the PepMV TGBp1 in yeast-two-hybrid screening, in vitro pull-down and bimolecular fluorescent complementation (BiFC) assays. SlTXND9 possesses part of the conserved thioredoxin (TRX) active site sequence (W__PC vs. WCXPC), and TXND9 orthologues cluster within the TRX phylogenetic superfamily closest to phosducin-like protein-3. In PepMV-infected and healthy Nicotiana benthamiana plants, NbTXND9 mRNA levels were comparable, and expression levels remained stable in both local and systemic leaves for 10 days post inoculation (dpi), as was also the case for catalase 1 (CAT1). To localize the TXND9 in plant cells, a polyclonal antiserum was produced. Purified α-SlTXND9 immunoglobulin (IgG) consistently detected a set of three protein bands in the range of 27–35 kDa, in the 1000 and 30,000 g pellets, and the soluble fraction of extracts of healthy and PepMV-infected N. benthamiana leaves, but not in the cell wall. These bands likely consist of the homologous protein NbTXND9 and its post-translationally modified derivatives. On electron microscopy, immuno-gold labelling of ultrathin sections of PepMV-infected N. benthamiana leaves using α-SlTXND9 IgG revealed particle accumulation close to plasmodesmata, suggesting a role in virus movement. Taken together, this study highlights a novel tomato-PepMV protein interaction and provides data on its localization in planta. Currently, studies focusing on the biological function of this interaction during PepMV infection are in progress.

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2000.13.5.520 ◽

2000 ◽

Vol 13 (5) ◽

pp. 520-528 ◽

Cited By ~ 59

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.

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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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.10.1158 ◽

2001 ◽

Vol 14 (10) ◽

pp. 1158-1167 ◽

Cited By ~ 63

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.

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