Beet necrotic yellow vein virus 42 kDa triple gene block protein binds nucleic acid in vitro

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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Molecular biology of potexviruses: recent advances

Journal of General Virology ◽

10.1099/vir.0.82667-0 ◽

2007 ◽

Vol 88 (6) ◽

pp. 1643-1655 ◽

Cited By ~ 109

Author(s):

Jeanmarie Verchot-Lubicz ◽

Chang-Ming Ye ◽

Devinka Bamunusinghe

Keyword(s):

Gene Silencing ◽

Rna Synthesis ◽

Triple Gene Block ◽

Potato Virus X ◽

Future Research ◽

Virus Movement ◽

Suppressor Activity ◽

Gene Block ◽

Recent Advances

Recent advances in potexvirus research have produced new models describing virus replication, cell-to-cell movement, encapsidation, R gene-mediated resistance and gene silencing. Interactions between distant RNA elements are a central theme in potexvirus replication. The 5′ non-translated region (NTR) regulates genomic and subgenomic RNA synthesis and encapsidation, as well as virus plasmodesmal transport. The 3′ NTR regulates both plus- and minus-strand RNA synthesis. How the triple gene-block proteins interact for virus movement is still elusive. As the potato virus X (PVX) TGBp1 protein gates plasmodesmata, regulates virus translation and is a suppressor of RNA silencing, further research is needed to determine how these properties contribute to propelling virus through the plasmodesmata. Specifically, TGBp1 suppressor activity is required for virus movement, but how the silencing machinery relates to plasmodesmata is not known. The TGBp2 and TGBp3 proteins are endoplasmic reticulum (ER)-associated proteins required for virus movement. TGBp2 associates with ER-derived vesicles that traffic along the actin network. Future research will determine whether the virus-induced vesicles are cytopathic structures regulating events along the ER or are vehicles carrying virus to the plasmodesmata for transfer into neighbouring cells. Efforts to assemble virions in vitro identified a single-tailed particle (STP) comprising RNA, coat protein (CP) and TGBp1. It has been proposed that TGBp1 aids in transport of virions or STP between cells and ensures translation of RNA in the receiving cells. PVX is also a tool for studying Avr–R gene interactions and gene silencing in plants. The PVX CP is the elicitor for the Rx gene. Recent reports of the PVX CP reveal how CP interacts with the Rx gene product.

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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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Complete Genome Sequence of aLily virus XIsolate from Japan

Genome Announcements ◽

10.1128/genomea.01462-17 ◽

2018 ◽

Vol 6 (4) ◽

Author(s):

Takamichi Nijo ◽

Yukari Okano ◽

Masayoshi Kondo ◽

Hiroaki Okuhara ◽

Hiroyo Sekimura ◽

...

Keyword(s):

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Triple Gene Block ◽

Start Codon ◽

Gene Block ◽

First Time ◽

Triple Gene Block Protein

ABSTRACTThe complete genome sequence ofLily virus X(LVX), which infects lilies, was determined for the first time from lilies in Japan. As with previous reports, the genome of the Japanese LVX isolate lacked an AUG start codon for the triple gene block protein 3-like region.

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Functional Analyses and Identification of Two Arginine Residues Essential to the ATP-Utilizing Activity of the Triple Gene Block Protein 1 of Bamboo Mosaic Potexvirus

Virology ◽

10.1006/viro.2000.0610 ◽

2000 ◽

Vol 277 (2) ◽

pp. 336-344 ◽

Cited By ~ 25

Author(s):

Dann-Ying Liou ◽

Yau-Heiu Hsu ◽

Chiung-Hua Wung ◽

Wen-Horng Wang ◽

Na-Sheng Lin ◽

...

Keyword(s):

Triple Gene Block ◽

Gene Block ◽

Arginine Residues ◽

Functional Analyses ◽

Triple Gene Block Protein

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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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