Phloem Unloading of Potato virus X Movement Proteins Is Regulated by Virus and Host Factors

To determine the requirements for viral proteins exiting the phloem, transgenic plants expressing green fluorescent protein (GFP) fused to the Potato virus X (PVX) triple gene block (TGB)p1 and coat protein (CP) genes were prepared. The fused genes were transgenically expressed from the companion cell (CC)-specific Commelina yellow mottle virus (CoYMV) promoter. Transgenic plants were selected for evidence of GFP fluorescence in CC and sieve elements (SE) and proteins were determined to be phloem mobile based on their ability to translocate across a graft union into nontransgenic scions. Petioles and leaves were analyzed to determine the requirements for phloem unloading of the fluorescence proteins. In petioles, fluorescence spread throughout the photosynthetic vascular cells (chlorenchyma) but did not move into the cortex, indicating a specific barrier to proteins exiting the vasculature. In leaves, fluorescence was mainly restricted to the veins. However, in virus-infected plants or leaves treated with a cocktail of proteasome inhibitors, fluorescence spread into leaf mesophyll cells. These data indicate that PVX contributes factors which enable specific unloading of cognate viral proteins and that proteolysis may play a role in limiting proteins in the phloem and surrounding chlorenchyma.

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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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Evidence that the Linker between the Methyltransferase and Helicase Domains of Potato Virus X Replicase Is Involved in Homologous RNA Recombination

Journal of Virology ◽

10.1128/jvi.00179-08 ◽

2009 ◽

Vol 83 (15) ◽

pp. 7761-7769 ◽

Cited By ~ 7

Author(s):

Heidrun-Katharina Draghici ◽

Mark Varrelmann

Keyword(s):

Coat Protein ◽

Potato Virus ◽

Fluorescent Protein ◽

Triple Gene Block ◽

Leaf Tissue ◽

Potato Virus X ◽

Wild Type Virus ◽

Rna Recombination ◽

Gene Block

ABSTRACT Recombination in RNA viruses, one of the main factors contributing to their genetic variability and evolution, is a widespread phenomenon. In this study, an in vivo assay to characterize RNA recombination in potato virus X (PVX), under high selection pressure, was established. Agrobacterium tumefaciens was used to express in Nicotiana benthamiana leaf tissue both a PVX isolate labeled with green fluorescent protein (GFP) containing a coat protein deletion mutation (ΔCP) and a transcript encoding a functional coat protein +3′-ntr. Coexpression of the constructs led to virus movement and systemic infection; reconstituted recombinants were observed in 92% of inoculated plants. Similar results were obtained using particle bombardment, demonstrating that recombination mediated by A. tumefaciens was not responsible for the occurrence of PXC recombinants. The speed of recombination could be estimated by agroinfection of two PVX mutants lacking the 3′ and 5′ halves of the genome, respectively, with an overlap in the triple gene block 1 gene, allowing GFP expression only in the case of recombination. Ten different pentapeptide insertion scanning replicase mutants with replication abilities comparable to wild-type virus were applied in the different recombination assays. Two neighboring mutants affecting the linker between the methyltransferase and helicase domains were shown to be strongly debilitated in their ability to recombine. The possible functional separation of replication and recombination in the replicase molecule supports the model that RNA recombination represents a distinct function of this protein, although the underlying mechanism still needs to be investigated.

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Expression Strategy of the Potato Virus X Triple Gene Block

Journal of General Virology ◽

10.1099/0022-1317-72-8-2039 ◽

1991 ◽

Vol 72 (8) ◽

pp. 2039-2042 ◽

Cited By ~ 33

Author(s):

S. Yu. Morozov ◽

N. A. Miroshnichenko ◽

A. G. Solovyev ◽

O. N. Fedorkin ◽

D. A. Zelenina ◽

...

Keyword(s):

Potato Virus ◽

Triple Gene Block ◽

Potato Virus X ◽

Gene Block

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Plant Translation Elongation Factor 1Bβ Facilitates Potato Virus X (PVX) Infection and Interacts with PVX Triple Gene Block Protein 1

PLoS ONE ◽

10.1371/journal.pone.0128014 ◽

2015 ◽

Vol 10 (5) ◽

pp. e0128014 ◽

Cited By ~ 12

Author(s):

JeeNa Hwang ◽

Seonhee Lee ◽

Joung-Ho Lee ◽

Won-Hee Kang ◽

Jin-Ho Kang ◽

...

Keyword(s):

Potato Virus ◽

Triple Gene Block ◽

Elongation Factor ◽

Potato Virus X ◽

Translation Elongation Factor ◽

Translation Elongation ◽

Gene Block ◽

Triple Gene Block Protein

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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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In Vivo Translation of the Triple Gene Block of Potato Virus X Requires Two Subgenomic mRNAs

Journal of Virology ◽

10.1128/jvi.72.10.8316-8320.1998 ◽

1998 ◽

Vol 72 (10) ◽

pp. 8316-8320 ◽

Cited By ~ 64

Author(s):

Jeanmarie Verchot ◽

Susan M. Angell ◽

David C. Baulcombe

Keyword(s):

Potato Virus ◽

Triple Gene Block ◽

Mrna Translation ◽

Potato Virus X ◽

Open Reading Frames ◽

Movement Proteins ◽

Gene Block ◽

Overlapping Open Reading Frames ◽

Reading Frames

ABSTRACT The 25-kilodalton (25K), 12K, and 8K movement proteins of potato virus X are derived from overlapping open reading frames (ORFs). Using an in vivo complementation assay, we have shown that the 25K protein is expressed from a functionally monocistronic mRNA, whereas the 12K and 8K proteins are from a bicistronic mRNA. Translation of the 8K ORF is by leaky ribosome scanning through the 12K ORF.

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2000.13.6.599 ◽

2000 ◽

Vol 13 (6) ◽

pp. 599-605 ◽

Cited By ~ 63

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.

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